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.
Project description:All cells must integrate sensory information to coordinate developmental events in space and time. The bacterium Caulobacter crescentus uses two-component phospho-signalling to regulate spatially distinct cell cycle events through the master regulator CtrA. Here, we report that CckA, the histidine kinase upstream of CtrA, employs a tandem-PAS domain sensor to integrate two distinct spatiotemporal signals. Using CckA reconstituted on liposomes, we show that one PAS domain modulates kinase activity in a CckA density-dependent manner, mimicking the stimulation of CckA kinase activity that occurs on its transition from diffuse to densely packed at the cell poles. The second PAS domain interacts with the asymmetrically partitioned second messenger cyclic-di-GMP, inhibiting kinase activity while stimulating phosphatase activity, consistent with the selective inactivation of CtrA in the incipient stalked cell compartment. The integration of these spatially and temporally regulated signalling events within a single signalling receptor enables robust orchestration of cell-type-specific gene regulation.
Project description:Sophisticated signaling mechanisms allow bacterial cells to cope with environmental and intracellular challenges. Activation of specific pathways ameliorates these challenges and thereby warrants integrity. Here, we demonstrate the pliability of the CckA-CtrA two-component signaling system in the freshwater bacterium Caulobacter crescentus. Our forward genetic screen to analyze suppressor mutations that can negate the chromosome segregation block induced by the topoisomerase IV inhibitor, NstA, yielded various point mutations in the cell cycle histidine kinase, CckA. Notably, we identified a point mutation in the PAS-B domain of CckA, which resulted in increased levels of phosphorylated CtrA (CtrA~P), the master cell cycle regulator. Surprisingly, this increase in CtrA~P levels did not translate into a genome-wide increase in the DNA occupancy of CtrA, but specifically enriched its affinity for the chromosomal origin of replication, Cori, and for a very small sub-set of CtrA regulated promoters. We show that through this enhanced binding of CtrA to the Cori, cells are able to overcome the toxic defects rendered by stable NstA through a possible slow down in the chromosome replication cycle. Taken together, our work opens up an unexplored and intriguing aspect of the CckA-CtrA signal transduction pathway. The distinctive DNA binding nature of CtrA and its regulation by CckA might also be crucial for pathogenesis because of the highly conserved nature of the CckA-CtrA pathway in alphaproteobacteria. Highlights • The PAS-B domain of the CckA histidine kinase modulates the distinctive DNA binding of CtrA in Caulobacter crescentus.• The DNA binding of CtrA at the Cori and at S-phase promoters is specifically enhanced due to a mutation in the PAS-B domain.• Plasticity in the CckA-CtrA pathway facilitates Caulobacter to overcome the NstA-induced chromosome segregation defect.
Project description:The bacterial cell cycle has been extensively studied under standard growth conditions. How it is modulated in response to environmental changes remains poorly understood. Here, we demonstrate that the freshwater bacterium Caulobacter crescentus blocks cell division and grows to filamentous cells in response to stress conditions affecting the cell membrane. Our data suggest that stress switches the membrane-bound cell cycle kinase CckA to its phosphatase mode, leading to the rapid dephosphorylation, inactivation and proteolysis of the master cell cycle regulator CtrA. The clearance of CtrA results in downregulation of division and morphogenesis genes and consequently a cell division block. Upon shift to non-stress conditions, cells quickly restart cell division and return to normal cell size. Our data indicate that the temporary inhibition of cell division through the regulated inactivation of CtrA constitutes a growth advantage under stress. Taken together, our work reveals a new mechanism that allows bacteria to alter their mode of proliferation in response to environmental cues by controlling the activity of a master cell cycle transcription factor. Furthermore, our results highlight the role of a bifunctional kinase in this process that integrates the cell cycle with environmental information.
Project description:Bacteria respond to their environment via signal transduction pathways, often two-component type systems that function through phosphotransfer to control expression of specific genes. Phosphorelays are derived from two-component systems but are comprised of additional components. The essential cckA-chpT-ctrA phosphorelay in Caulobacter crescentus has been well studied and is important in orchestrating the cell cycle, polar development and flagellar biogenesis. Although cckA, chpT and ctrA homologues are widespread among the Alphaproteobacteria, relatively few is known about their function in the large and ecologically significant Roseobacter clade of the Rhodobacterales. In this study the cckA-chpT-ctrA system of the marine sponge symbiont Ruegeria sp. KLH11 was investigated. Our results reveal that the cckA, chpT and ctrA genes positively control flagellar biosynthesis. In contrast to C. crescentus, the cckA, chpT and ctrA genes in Ruegeria sp. KLH11 are non-essential and do not affect bacterial growth. Gene fusion and transcript analyses provide evidence for ctrA autoregulation and the control of motility-related genes. In KLH11, flagellar motility is controlled by the SsaRI system and acylhomoserine lactone (AHL) quorum sensing. SsaR and long chain AHLs are required for cckA, chpT and ctrA gene expression, providing a regulatory link between flagellar locomotion and population density in KLH11.
Project description:A core regulatory pathway that directs developmental transitions and cellular asymmetries in Agrobacterium tumefaciens involves two overlapping, integrated phosphorelays. One of these phosphorelays putatively includes four histidine sensor kinase homologues, DivJ, PleC, PdhS1 and PdhS2, and two response regulators, DivK and PleD. In several different alphaproteobacteria, this pathway influences a conserved downstream phosphorelay that ultimately controls the phosphorylation state of the CtrA master response regulator. The PdhS2 sensor kinase reciprocally regulates biofilm formation and swimming motility. In the current study, the mechanisms by which the A. tumefaciens sensor kinase PdhS2 directs this regulation are delineated. PdhS2 lacking a key residue implicated in phosphatase activity is markedly deficient in proper control of attachment and motility phenotypes, whereas a kinase-deficient PdhS2 mutant is only modestly affected. A genetic interaction between DivK and PdhS2 is revealed, unmasking one of several connections between PdhS2-dependent phenotypes and transcriptional control by CtrA. Epistasis experiments suggest that PdhS2 may function independently of the CckA sensor kinase, the cognate sensor kinase for CtrA, which is inhibited by DivK. Global expression analysis of the pdhS2 mutant reveals a restricted regulon, most likely functioning through CtrA to separately control motility and regulate the levels of the intracellular signal cyclic diguanylate monophosphate (cdGMP), thereby affecting the production of adhesive polysaccharides and attachment. We hypothesize that in A. tumefaciens the CtrA regulatory circuit has expanded to include additional inputs through the addition of PdhS-type sensor kinases, likely fine-tuning the response of this organism to the soil microenvironment.
Project description:Cell-fate asymmetry in the predivisional cell of Caulobacter crescentus requires that the regulatory protein DivL localizes to the new pole of the cell where it up-regulates CckA kinase, resulting in a gradient of CtrA~P across the cell. In the preceding stage of the cell cycle (the "stalked" cell), DivL is localized uniformly along the cell membrane and maintained in an inactive form by DivK~P. It is unclear how DivL overcomes inhibition by DivK~P in the predivisional cell simply by changing its location to the new pole. It has been suggested that co-localization of DivL with PleC phosphatase at the new pole is essential to DivL's activity there. However, there are contrasting views on whether the bifunctional enzyme, PleC, acts as a kinase or phosphatase at the new pole. To explore these ambiguities, we formulated a mathematical model of the spatiotemporal distributions of DivL, PleC and associated proteins (DivJ, DivK, CckA, and CtrA) during the asymmetric division cycle of a Caulobacter cell. By varying localization profiles of DivL and PleC in our model, we show how the physiologically observed spatial distributions of these proteins are essential for the transition from a stalked cell to a predivisional cell. Our simulations suggest that PleC is a kinase in predivisional cells, and that, by sequestering DivK~P, the kinase form of PleC enables DivL to be reactivated at the new pole. Hence, co-localization of PleC kinase and DivL is essential to establishing cellular asymmetry. Our simulations reproduce the experimentally observed spatial distribution and phosphorylation status of CtrA in wild-type and mutant cells. Based on the model, we explore novel combinations of mutant alleles, making predictions that can be tested experimentally.
Project description:Caulobacter crescentus initiates a single round of DNA replication during each cell cycle. Following the initiation of DNA replication, the essential CckA histidine kinase is activated by phosphorylation, which (via the ChpT phosphotransferase) enables the phosphorylation and activation of the CtrA global regulator. CtrA approximately P then blocks the reinitiation of replication while regulating the transcription of a large number of cell cycle-controlled genes. It has been shown that DNA replication serves as a checkpoint for flagellar biosynthesis and cell division and that this checkpoint is mediated by the availability of active CtrA. Because CckA approximately P promotes the activation of CtrA, we addressed the question of what controls the temporal activation of CckA. We found that the initiation of DNA replication is a prerequisite for remodeling the new cell pole, which includes the localization of the DivL protein kinase to that pole and, consequently, the localization, autophosphorylation, and activation of CckA at that pole. Thus, CckA activation is dependent on polar remodeling and a DNA replication initiation checkpoint that is tightly integrated with the polar phospho-signaling cascade governing cell cycle progression.
Project description:The ?-Proteobacterium Agrobacterium tumefaciens has proteins homologous to known regulators that govern cell division and development in Caulobacter crescentus, many of which are also conserved among diverse ?-Proteobacteria. In light of recent work demonstrating similarity between the division cycle of C. crescentus and that of A. tumefaciens, the functional conservation for this presumptive control pathway was examined. In C. crescentus the CtrA response regulator serves as the master regulator of cell cycle progression and cell division. CtrA activity is controlled by an integrated pair of multi-component phosphorelays: PleC/DivJ-DivK and CckA-ChpT-CtrA. Although several of the conserved orthologues appear to be essential in A. tumefaciens, deletions in pleC or divK were isolated and resulted in cell division defects, diminished swimming motility, and a decrease in biofilm formation. A. tumefaciens also has two additional pleC/divJhomologue sensor kinases called pdhS1 and pdhS2, absent in C. crescentus. Deletion of pdhS1 phenocopied the ?pleC and ?divK mutants. Cells lacking pdhS2 morphologically resembled wild-type bacteria, but were decreased in swimming motility and elevated for biofilm formation, suggesting that pdhS2 may serve to regulate the motile to non-motile switch in A. tumefaciens. Genetic analysis suggests that the PleC/DivJ-DivK and CckA-ChpT-CtrA phosphorelays in A. tumefaciens are vertically-integrated, as in C. crescentus. A gain-of-function mutation in CckA (Y674D) was identified as a spontaneous suppressor of the ?pleC motility phenotype. Thus, although the core architecture of the A. tumefaciens pathway resembles that of C. crescentus there are specific differences including additional regulators, divergent pathway architecture, and distinct target functions.
Project description:The phosphorylated form of the response regulator CtrA represses DNA replication initiation and regulates the transcription of about 100 cell cycle-regulated genes in Caulobacter crescentus. CtrA activity fluctuates during the cell cycle, and its periodicity is a key element of the engine that drives cell cycle progression. The histidine kinase CckA controls the phosphorylation not only of CtrA but also of CpdR, whose unphosphorylated form promotes CtrA proteolysis. Thus, CckA has a central role in establishing the cell cycle periodicity of CtrA activity by controlling both its phosphorylation and stability. Evidence suggests that the polar localization of CckA during the cell cycle plays a role in CckA function. However, the exact pattern of CckA localization remains controversial. Here, we describe a thorough, quantitative analysis of the spatiotemporal distribution of a functional and chromosomally produced CckA-monomeric green fluorescent protein fusion that affects current models of cell cycle regulation. We also identify two cis-acting regions in CckA that are important for its proper localization and function. The disruption of a PAS-like motif in the sensor domain affects the stability of CckA accumulation at the poles. This is accompanied by a partial loss in CckA function. Shortening an extended linker between beta-sheets within the CckA catalysis-assisting ATP-binding domain has a more severe effect on CckA polar localization and function. This mutant strain exhibits a dramatic cell-to-cell variability in CpdR levels and CtrA cell cycle periodicity, suggesting that the cell cycle-coordinated polar localization of CckA may be important for the robustness of signal transduction and cell cycle progression.
Project description:Histidine kinases are key components of regulatory networks in bacteria. Although many of these enzymes are bifunctional, mediating both phosphorylation and dephosphorylation of downstream targets, the molecular details of this central regulatory switch are unclear. We showed recently that the universal second messenger cyclic di-guanosine monophosphate (c-di-GMP) drives Caulobacter crescentus cell cycle progression by forcing the cell cycle kinase CckA from its default kinase into phosphatase mode. We use a combination of structure determination, modeling, and functional analysis to demonstrate that c-di-GMP reciprocally regulates the two antagonistic CckA activities through noncovalent cross-linking of the catalytic domain with the dimerization histidine phosphotransfer (DHp) domain. We demonstrate that both c-di-GMP and ADP (adenosine diphosphate) promote phosphatase activity and propose that c-di-GMP stabilizes the ADP-bound quaternary structure, which allows the receiver domain to access the dimeric DHp stem for dephosphorylation. In silico analyses predict that c-di-GMP control is widespread among bacterial histidine kinases, arguing that it can replace or modulate canonical transmembrane signaling.