A dynamically localized protease complex and a polar specificity factor control a cell cycle master regulator

Regulated proteolysis is essential for cell cycle progression in both prokaryotes and eukaryotes. We show here that the ClpXP protease, responsible for the degradation of multiple bacterial proteins, is dynamically localized to specific cellular positions in Caulobacter where it degrades colocalized proteins. The CtrA cell cycle master regulator, that must be cleared from the Caulobacter cell to allow the initiation of chromosome replication, interacts with the ClpXP protease at the cell pole where it is degraded. We have identified a novel, conserved protein, RcdA, that forms a complex with CtrA and ClpX in the cell. RcdA is required for CtrA polar localization and degradation by ClpXP. The localization pattern of RcdA is coincident with and dependent upon ClpX localization. Thus, a dynamically localized ClpXP proteolysis complex in concert with a cytoplasmic factor provides temporal and spatial specificity to protein degradation during a bacterial cell cycle.     

Activation and polar sequestration of PopA,a c‐di‐GMP effector protein involved in Caulobacter crescentus cell cycle control

When Caulobacter crescentus enters S‐phase the replication initiation inhibitor CtrA dynamically positions to the old cell pole to be degraded by the polar ClpXP protease. Polar delivery of CtrA requires PopA and the diguanylate cyclase PleD that positions to the same pole. Here we present evidence that PopA originated through gene duplication from its paralogue response regulator PleD and subsequent co‐option as c‐di‐GMP effector protein. While the C‐terminal catalytic domain (GGDEF) of PleD is activated by phosphorylation of the N‐terminal receiver domain, functional adaptation has reversed signal transduction in PopA with the GGDEF domain adopting input function and the receiver domain serving as regulatory output. We show that the N‐terminal receiver domain of PopA specifically interacts with RcdA, a component required for CtrA degradation. In contrast, the GGDEF domain serves to target PopA to the cell pole in response to c‐di‐GMP binding. In agreement with the divergent activation and targeting mechanisms, distinct markers sequester PleD and PopA to the old cell pole upon S‐phase entry. Together these data indicate that PopA adopted a novel role as topology specificity factor to help recruit components of the CtrA degradation pathway to the protease specific old cell pole of C. crescentus.     

Regulated proteolysis of a transcription factor complex is critical to cell cycle progression in Caulobacter crescentus

Cell cycle transitions are often triggered by the proteolysis of key regulatory proteins. In Caulobacter crescentus, the G1‐S transition involves the degradation of an essential DNA‐binding response regulator, CtrA, by the ClpXP protease. Here, we show that another critical cell cycle regulator, SciP, is also degraded during the G1‐S transition, but by the Lon protease. SciP is a small protein that binds directly to CtrA and prevents it from activating target genes during G1. We demonstrate that SciP must be degraded during the G1‐S transition so that cells can properly activate CtrA‐dependent genes following DNA replication initiation and the reaccumulation of CtrA. These results indicate that like CtrA, SciP levels are tightly regulated during the Caulobacter cell cycle. In addition, we show that formation of a complex between CtrA and SciP at target promoters protects both proteins from their respective proteases. Degradation of either protein thus helps trigger the destruction of the other, facilitating a cooperative disassembly of the complex. Collectively, our results indicate that ClpXP and Lon each degrade an important cell cycle regulator, helping to trigger the onset of S phase and prepare cells for the subsequent programmes of gene expression critical to polar morphogenesis and cell division.     

An essential protease involved in bacterial cell-cycle control.

Proteolytic inactivation of key regulatory proteins is essential in eukaryotic cell-cycle control. We have identified a protease in the eubacterium Caulobacter crescentus that is indispensable for viability and cell-cycle progression, indicating that proteolysis is also involved in controlling the bacterial cell cycle. Mutants of Caulobacter that lack the ATP-dependent serine protease ClpXP are arrested in the cell cycle before the initiation of chromosome replication and are blocked in the cell division process. ClpXP is composed of two types of polypeptides, the ClpX ATPase and the ClpP peptidase. Site-directed mutagenesis of the catalytically active serine residue of ClpP confirmed that the proteolytic activity of ClpXP is essential. Analysis of mutants lacking ClpX or ClpP revealed that both proteins are required in vivo for the cell-cycle-dependent degradation of the regulatory protein CtrA. CtrA is a member of the response regulator family of two-component signal transduction systems and controls multiple cell-cycle processes in Caulobacter. In particular, CtrA negatively controls DNA replication and our findings suggest that specific degradation of the CtrA protein by the ClpXP protease contributes to G1-to-S transition in this organism.     

Sinorhizobium meliloti CpdR1 is critical for co-ordinating cell cycle progression and the symbiotic chronic infection

ATP-driven proteolysis plays a major role in regulating the bacterial cell cycle, development and stress responses. In the nitro -fixing symbiosis with host plants, Sinorhizobium meliloti undergoes a profound cellular differentiation, including endoreduplication of the ome. The regulatory mechanisms governing the alterations of the S. meliloti cell cycle in planta are largely unknown. Here, we report the characterization of two cpdR homologues, cpdR1 and cpdR2 , of S. meliloti that encode single-domain response regulators. In Caulobacter crescentus , CpdR controls the polar localization of the ClpXP protease, thereby mediating the regulated proteolysis of key protein(s), such as CtrA, involved in cell cycle progression. The S. meliloti cpdR1 -null mutant can invade the host cytoplasm, however, the intracellular bacteria are unable to differentiate into bacteroids. We show that S. meliloti CpdR1 has a polar localization pattern and a role in ClpX positioning similar to C. crescentus CpdR, suggesting a conserved function of CpdR proteins among α-proteobacteria. However, in S. meliloti , free-living cells of the cpdR1 -null mutant show a striking morphology of irregular coccoids and aberrant DNA replication. Thus, we demonstrate that CpdR1 mediates the co-ordination of cell cycle events, which are critical for both the free-living cell division and the differentiation required for the chronic intracellular infection.     

Turnover of endogenous SsrA-tagged proteins mediated by ATP-dependent proteases in Escherichia coli

Formation and degradation of SsrA-tagged proteins enable ribosome recycling and elimination of defective products of incomplete translation. We produced an antibody against the SsrA peptide and used it to measure the amounts of SsrA-tagged proteins in Escherichia coli cells without interfering with tagging or altering the context of the tag added at the ends of nascent polypeptides. SsrA-tagged proteins were present in very small amounts unless a component of the ClpXP protease was missing. From the levels of tagged proteins in cells in which degradation is essentially blocked, we calculate that > or =1 in 200 translation products receives an SsrA tag. ClpXP is responsible for > or =90% of the degradation of SsrA-tagged proteins. The degradation rate in wild type cells is > or =1.4 min(-1) and decreases to approximately 0.10 min(-1) in a clpX mutant. The rate of degradation by ClpXP is decreased approximately 3-fold in mutants lacking the adaptor SspB, whereas degradation by ClpAP is increased 3-5-fold. However, ClpAP degrades SsrA-tagged proteins slowly even in the absence of SspB, possibly because of interference from ClpA-specific substrates. Lon protease degrades SsrA-tagged proteins at a rate of approximately 0.05 min(-1) in the presence or absence of SspB. We conclude that ClpXP, together with SspB, is uniquely adapted for degradation of SsrA-tagged proteins and is responsible for the major part of their degradation in vivo.     

Regulated degradation of chromosome replication proteins DnaA and CtrA in Caulobacter crescentus

DnaA protein binds bacterial replication origins and it initiates chromosome replication. The Caulobacter crescentus DnaA also initiates chromosome replication and the C. crescentus response regulator CtrA represses chromosome replication. CtrA proteolysis by ClpXP helps restrict chromosome replication to the dividing cell type. We report that C. crescentus DnaA protein is also selectively targeted for proteolysis but DnaA proteolysis uses a different mechanism. DnaA protein is unstable during both growth and stationary phases. During growth phase, DnaA proteolysis ensures that primarily newly made DnaA protein is present at the start of each replication period. Upon entry into stationary phase, DnaA protein is completely removed while CtrA protein is retained. Cell cycle arrest by sudden carbon or nitrogen starvation is sufficient to increase DnaA proteolysis, and relieving starvation rapidly stabilizes DnaA protein. This starvation-induced proteolysis completely removes DnaA protein even while DnaA synthesis continues. Apparently, C. crescentus relies on proteolysis to adjust DnaA in response to such rapid nutritional changes. Depleting the C. crescentus ClpP protease significantly stabilizes DnaA. However, a dominant-negative clpX allele that blocks CtrA degradation, even when combined with a clpA null allele, did not decrease DnaA degradation. We suggest that either a novel chaperone presents DnaA to ClpP or that ClpX is used with exceptional efficiency so that when ClpX activity is limiting for CtrA degradation it is not limiting for DnaA degradation. This unexpected and finely tuned proteolysis system may be an important adaptation for a developmental bacterium that is often challenged by nutrient-poor environments.     

Cell cycle-dependent polar localization of an essential bacterial histidine kinase that controls DNA replication and cell division

The master CtrA response regulator functions in Caulobacter to repress replication initiation in different phases of the cell cycle. Here, we identify an essential histidine kinase, CckA, that is responsible for CtrA activation by phosphorylation. Although CckA is present throughout the cell cycle, it moves to a cell pole in S phase, and upon cell division it disperses. Removal of the membrane-spanning region of CckA results in loss of polar localization and cell death. We propose that polar CckA functions to activate CtrA just after the initiation of DNA replication, thereby preventing premature reinitiations of chromosome replication. Thus, dynamic changes in cellular location of critical signal proteins provide a novel mechanism for the control of the prokaryote cell cycle.     

SpbR overproduction reveals the importance of proteolytic degradation for cell pole development and chromosome segregation in Caulobacter crescentus

In most rod‐shaped bacteria, DNA replication is quickly followed by chromosome segregation, when one of the newly duplicated centromeres moves across the cell to the opposite (or ‘new’) pole. Two proteins in Caulobacter crescentus, PopZ and TipN, provide directional cues at the new pole that guide the translocating chromosome to its destination. We show that centromere translocation can be inhibited by an evolutionarily conserved pole‐localized protein that we have named SpbR. When overproduced, SpbR exhibits aberrant accumulation at the old pole, where it physically interacts with PopZ. This prevents the relocation of PopZ to the new pole, thereby eliminating a positional cue for centromere translocation. Consistent with this, the centromere translocation phenotype of SpbR overproducing cells is strongly enhanced in a ?tipN mutant background. We find that pole‐localized SpbR is normally cleared by ClpXP‐mediated proteolysis before the time of chromosome segregation, indicating that SpbR turnover is part of the cell cycle‐dependent program of polar development. This work demonstrates the importance of proteolysis as a housekeeping activity that removes outgoing factors from the developing cell pole, and provides an example of a substrate that can inhibit polar functions if it is insufficiently cleared.     

Modeling the temporal dynamics of master regulators and CtrA proteolysis in Caulobacter crescentus cell cycle

The cell cycle of Caulobacter crescentus involves the polar morphogenesis and an asymmetric cell division driven by precise interactions and regulations of proteins, which makes Caulobacter an ideal model organism for investigating bacterial cell development and differentiation. The abundance of molecular data accumulated on Caulobacter motivates system biologists to analyze the complex regulatory network of cell cycle via quantitative modeling. In this paper, We propose a comprehensive model to accurately characterize the underlying mechanisms of cell cycle regulation based on the study of: a) chromosome replication and methylation; b) interactive pathways of five master regulatory proteins including DnaA, GcrA, CcrM, CtrA, and SciP, as well as novel consideration of their corresponding mRNAs; c) cell cycle-dependent proteolysis of CtrA through hierarchical protease complexes. The temporal dynamics of our simulation results are able to closely replicate an extensive set of experimental observations and capture the main phenotype of seven mutant strains of Caulobacter crescentus. Collectively, the proposed model can be used to predict phenotypes of other mutant cases, especially for nonviable strains which are hard to cultivate and observe. Moreover, the module of cyclic proteolysis is an efficient tool to study the metabolism of proteins with similar mechanisms.     

Cell Cycle Control by the Master Regulator CtrA in Sinorhizobium meliloti

In all domains of life, proper regulation of the cell cycle is critical to coordinate genome replication, segregation and cell division. In some groups of bacteria, e.g. Alphaproteobacteria, tight regulation of the cell cycle is also necessary for the morphological and functional differentiation of cells. Sinorhizobium meliloti is an alphaproteobacterium that forms an economically and ecologically important nitrogen-fixing symbiosis with specific legume hosts. During this symbiosis S. meliloti undergoes an elaborate cellular differentiation within host root cells. The differentiation of S. meliloti results in massive amplification of the genome, cell branching and/or elongation, and loss of reproductive capacity. In Caulobacter crescentus, cellular differentiation is tightly linked to the cell cycle via the activity of the master regulator CtrA, and recent research in S. meliloti suggests that CtrA might also be key to cellular differentiation during symbiosis. However, the regulatory circuit driving cell cycle progression in S. meliloti is not well characterized in both the free-living and symbiotic state. Here, we investigated the regulation and function of CtrA in S. meliloti. We demonstrated that depletion of CtrA cause cell elongation, branching and genome amplification, similar to that observed in nitrogen-fixing bacteroids. We also showed that the cell cycle regulated proteolytic degradation of CtrA is essential in S. meliloti, suggesting a possible mechanism of CtrA depletion in differentiated bacteroids. Using a combination of ChIP-Seq and gene expression microarray analysis we found that although S. meliloti CtrA regulates similar processes as C. crescentus CtrA, it does so through different target genes. For example, our data suggest that CtrA does not control the expression of the Fts complex to control the timing of cell division during the cell cycle, but instead it negatively regulates the septum-inhibiting Min system. Our findings provide valuable insight into how highly conserved genetic networks can evolve, possibly to fit the diverse lifestyles of different bacteria.