Identification of a novel response regulator required for the swarmer-to-stalked-cell transition in Caulobacter crescentus.

The onset of motility late in the Caulobacter crescentus cell cycle depends on a signal transduction pathway mediated by the histidine kinase PleC and response regulator DivK. We now show that pleD, whose function is required for the subsequent loss of motility and stalk formation by the motile swarmer cell, encodes a 454-residue protein with tandem N-terminal response regulator domains D1 and D2 and a novel C-terminal GGDEF domain. The identification of pleD301, a semidominant suppressor of the pleC Mot phenotype, as a mutation predicted to result in a D-53-->G change in the D1 domain supports a role for phosphorylation in the PleD regulator. Disruptions constructed in the pleD open reading frame demonstrated that the gene is not essential and that the pleC phenotype can also be suppressed by a recessive, loss-of-function mutation. These results suggest that PleD is part of a signal transduction pathway controlling stalked-cell differentiation early in the C. crescentus cell cycle.     

Roles of the histidine protein kinase pleC in Caulobacter crescentus motility and chemotaxis.

The Caulobacter crescentus histidine kinase genes pleC and divJ have been implicated in the regulation of polar morphogenesis and cell division, respectively. Mutations in pleC also potentiate the cell division phenotype of divJ mutations. To investigate the involvement of the PleC kinase in motility and cell cycle regulation, we carried out a pseudoreversion analysis of the divJ332 allele, which confers a temperature-sensitive motility (Mot-) phenotype. All cold-sensitive pseudorevertants with a Mot+ phenotype at 37 degrees C and a cold-sensitive swarm phenotype in soft agar at 24 degrees C contained extragenic suppressors that were null mutations mapping to pleC. Instead of a cell division defect at the nonpermissive temperature, however, revertants displayed a cold-sensitive defect in chemotaxis (Che-). In addition, the mutant cells were also supermotile, a phenotype previously associated only with mutations in the response regulator gene pleD that block the loss of motility. We also found that the Mot- defect of pleC mutants is suppressed by a pleD301/pleD+ merodiploid and results in a similar, supermotile, cold-sensitive Che- phenotype. These results implicate signal transduction pathways mediated by PleC-DivK and DivJ-PleD in the regulation of chemotaxis as well as motility. We discuss these findings and the observation that although the PleC kinase does not play an indispensable role in cell division, a temperature-sensitive allele of pleC (pleC319) has severely reduced viability under stringent growth conditions.     

Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus

Several members of the two-component signal transduction family have been implicated in the control of polar development in Caulobacter crescentus: PleC and DivJ, two polarly localized histidine sensor kinases; and the response regulators DivK and PleD. The PleD protein was shown previously to be required during the swarmer-to-stalked cell transition for flagellar ejection and efficient stalk biogenesis. Here, we present data indicating that PleD also controls the onset of motility and a cell density switch immediately preceding cell division. Constitutively active alleles of pleD or wspR, an orthologue from Pseudomonas fluorescens, almost completely suppressed C. crescentus motility and inhibited the increase in swarmer cell density during cell differentiation. The observation that these alleles also had a dominant-negative effect on motility in a pleC divJ and a pleC divK mutant background indicated that PleD is located downstream of the other components in the signal transduction cascade, which controls the activity of the flagellar motor. In addition, the presence of a constitutive pleD or wspR allele resulted in a doubling of the average stalk length. Together, this is consistent with a model in which the active form of PleD, PleD approximately P, negatively controls aspects of differentiation in the late predivisional cell, whereas it acts positively on polar development during the swarmer-to-stalked cell transition. In agreement with such a model, we found that DivJ, which localizes to the stalked pole during cell differentiation, positively controlled the in vivo phosphorylation status of PleD, and the swarmer pole-specific PleC kinase modulated this status in a negative manner. Furthermore, domain switch experiments demonstrated that the WspR GGDEF output domain from P. fluorescens is active in C. crescentus, favouring a more general function for this novel signalling domain over a specific role such as DNA or protein interaction. Possible roles for PleD and its C-terminal output domain in modulating the polar cell surface of C. crescentus are discussed.     

Turning off flagellum rotation requires the pleiotropic gene pleD: pleA, pleC, and pleD define two morphogenic pathways in Caulobacter crescentus.

We have identified mutations in three pleiotropic genes, pleA, pleC, and pleD, that are required for differentiation in Caulobacter crescentus. pleA and pleC mutants were isolated in an extensive screen for strains defective in both motility and adsorption of polar bacteriophage phi CbK; using temperature-sensitive alleles, we determined the time at which the two genes act. pleA was required for a short period at 0.7 of the swarmer cell cycle for flagellum biosynthesis, whereas pleC was required during an overlapping period from 0.6 to 0.95 of the cell cycle to activate flagellum rotation as well as to enable loss of the flagellum and stalk formation by swarmer cells after division. The third pleiotropic gene, pleD, is described here for the first time. A pleD mutation was identified as a bypass suppressor of a temperature-sensitive pleC allele. Strains containing this mutation were highly motile, did not shed the flagellum or form stalks, and retained motility throughout the cell cycle. Since pleD was required to turn off motility and was a bypass suppressor of pleC, we conclude that it acts after the pleA and pleC gene functions in the cell cycle. No mutants defective in both flagellum biosynthesis and stalk formation were identified. Consequently, we propose that the steps required for formation of swarmer cells and subsequent development into stalked cells are organized into at least two developmental pathways: a pleA-dependent sequence of events, responsible for flagellum biosynthesis in predivisional cells, and a pleC-pleD-dependent sequence, responsible for flagellum activation in predivisional cells and loss of motility and stalk formation in progeny swarmer cells.     

An essential single domain response regulator required for normal cell division and differentiation in Caulobacter crescentus.

Signal transduction pathways mediated by sensor histidine kinases and cognate response regulators control a variety of physiological processes in response to environmental conditions. Here we show that in Caulobacter crescentus these systems also play essential roles in the regulation of polar morphogenesis and cell division. Previous studies have implicated histidine kinase genes pleC and divJ in the regulation of these developmental events. We now report that divK encodes an essential, cell cycle-regulated homolog of the CheY/Spo0F subfamily and present evidence that this protein is a cognate response regulator of the histidine kinase PleC. The purified kinase domain of PleC, like that of DivJ, can serve as an efficient phosphodonor to DivK and as a phospho-DivK phosphatase. Based on these and earlier genetic results we propose that PleC and DivK are members of a signal transduction pathway that couples motility and stalk formation to completion of a late cell division cycle event. Gene disruption experiments and the filamentous phenotype of the conditional divK341 mutant reveal that DivK also functions in an essential signal transduction pathway required for cell division, apparently in response to another histidine kinase. We suggest that phosphotransfer mediated by these two-component signal transduction systems may represent a general mechanism regulating cell differentiation and cell division in response to successive cell cycle checkpoints.     

Differential localization of two histidine kinases controlling bacterial cell differentiation

The bacterium C. crescentus coordinates cellular differentiation and cell cycle progression via a network of signal transduction proteins. Here, we demonstrate that the antagonistic DivJ and PleC histidine kinases that regulate polar differentiation are differentially localized as a function of the cell cycle. The DivJ kinase localizes to the stalked pole in response to a signal at the G1-to-S transition, while the PleC kinase is localized to the flagellar pole in swarmer and predivisional cells but is dispersed throughout the cell in the stalked cell. PleC, which is required for DivJ localization, may provide the cue at the G1-to-S transition that directs the polar positioning of DivJ. The dynamic positioning of signal transduction proteins may contribute to the regulation of polar differentiation at specific times during the bacterial cell cycle.     

The asymmetric spatial distribution of bacterial signal transduction proteins coordinates cell cycle events

The polar localization of signaling proteins that are essential for Caulobacter cell cycle control is temporally regulated. Here we provide evidence that phosphorylation of the essential response regulator, DivK, is required for both its function and its cell cycle-regulated localization. The asymmetric location of the DivJ and PleC histidine kinases and their antagonistic activities on the cellular concentration of phosphorylated DivK provide positional and temporal information for the ordered sequence of DivK localization during the cell cycle. DivJ activity on DivK affects its correct localization, which, in turn, is required for PleC function. Since DivJ and PleC regulate different cell cycle events, the interconnected function of these two histidine kinases through localization of a common response regulator provides a mechanism for coordinating cell cycle progression. Study of a DivK homolog in the morphologically symmetric bacterium Sinorhizobium meliloti suggests that this type of cell cycle mechanism is widespread in prokaryotes.     

Pseudoreversion Analysis Indicates a Direct Role of Cell Division Genes in Polar Morphogenesis and Differentiation in Caulobacter Crescentus

A pseudoreversion analysis was used to examine the role of cell division genes in polar morphogenesis in Caulobacter crescentus. Extragenic suppressors of temperature sensitive mutations in pleC, a pleiotropic gene required for cell motility, formation of polar phi CbK bacteriophage receptors, and stalk formation, were isolated. These suppressors, which restored motility at 37 degrees C, simultaneously conferred a cold sensitive cell division phenotype and they were mapped to the three new cell division genes divJ, divL and divK. The cold-sensitive mutations in divL, and to a lesser extent divJ, exhibited a relatively narrow range of suppression. The cold-sensitive cell division mutation in divK, by contrast, suppressed all pleC mutations examined and behaved as a classical bypass suppressor. The direct role of this cell division gene in the regulation of motility is suggested by the observation that divK341 mapped to the same locus as pleD301, a pleiotropic mutation that prevents loss of motility and stalk formation. These results provide strong evidence that the cell division and developmental pathways are interconnected and they support our earlier conclusion that cell division is required for the regulation of polar morphogenesis and differentiation in C. crescentus.     

Dynamical Localization of DivL and PleC in the Asymmetric Division Cycle of Caulobacter crescentus: A Theoretical Investigation of Alternative Models

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.     

The core dimerization domains of histidine kinases contain recognition specificity for the cognate response regulator

Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the Caulobacter crescentus cell cycle, respectively. The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC. We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK. The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily. The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases. A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer. We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases. The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO. We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.     

Interplay between the Localization and Kinetics of Phosphorylation in Flagellar Pole Development of the Bacterium Caulobacter crescentus

Bacterial cells maintain sophisticated levels of intracellular organization that allow for signal amplification, response to stimuli, cell division, and many other critical processes. The mechanisms underlying localization and their contribution to fitness have been difficult to uncover, due to the often challenging task of creating mutants with systematically perturbed localization but normal enzymatic activity, and the lack of quantitative models through which to interpret subtle phenotypic changes. Focusing on the model bacterium Caulobacter crescentus, which generates two different types of daughter cells from an underlying asymmetric distribution of protein phosphorylation, we use mathematical modeling to investigate the contribution of the localization of histidine kinases to the establishment of cellular asymmetry and subsequent developmental outcomes. We use existing mutant phenotypes and fluorescence data to parameterize a reaction-diffusion model of the kinases PleC and DivJ and their cognate response regulator DivK. We then present a systematic computational analysis of the effects of changes in protein localization and abundance to determine whether PleC localization is required for correct developmental timing in Caulobacter. Our model predicts the developmental phenotypes of several localization mutants, and suggests that a novel strain with co-localization of PleC and DivJ could provide quantitative insight into the signaling threshold required for flagellar pole development. Our analysis indicates that normal development can be maintained through a wide range of localization phenotypes, and that developmental defects due to changes in PleC localization can be rescued by increased PleC expression. We also show that the system is remarkably robust to perturbation of the kinetic parameters, and while the localization of either PleC or DivJ is required for asymmetric development, the delocalization of one of these two components does not prevent flagellar pole development. We further find that allosteric regulation of PleC observed in vitro does not affect the predicted in vivo developmental phenotypes. Taken together, our model suggests that cells can tolerate perturbations to localization phenotypes, whose evolutionary origins may be connected with reducing protein expression or with decoupling pre- and post-division phenotypes.     

Cytokinesis monitoring during development; rapid pole-to-pole shuttling of a signaling protein by localized kinase and phosphatase in Caulobacter

For successful generation of different cell types by asymmetric cell division, cell differentiation should be initiated only after completion of division. Here, we describe a control mechanism by which Caulobacter couples the initiation of a developmental program to the completion of cytokinesis. Genetic evidence indicates that localization of the signaling protein DivK at the flagellated pole prevents premature initiation of development. Photobleaching and FRET experiments show that polar localization of DivK is dynamic with rapid pole-to-pole shuttling of diffusible DivK generated by the localized activities of PleC phosphatase and DivJ kinase at opposite poles. This shuttling is interrupted upon completion of cytokinesis by the segregation of PleC and DivJ to different daughter cells, resulting in disruption of DivK localization at the flagellated pole and subsequent initiation of development in the flagellated progeny. Thus, dynamic polar localization of a diffusible protein provides a control mechanism that monitors cytokinesis to regulate development.     

The DivJ,CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti

Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen‐fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis.     

The Caulobacter crescentus polar organelle development protein PodJ is differentially localized and is required for polar targeting of the PleC development regulator

Regulation of polar development and cell division in Caulobacter crescentus relies on the dynamic localization of several proteins to cell poles at specific stages of the cell cycle. The polar organelle development protein, PodJ, is required for the synthesis of the adhesive holdfast and pili. Here we show the cell cycle localization of PodJ and describe a novel role for this protein in controlling the dynamic localization of the developmental regulator PleC. In swarmer cells, a short form of PodJ is localized at the flagellated pole. Upon differentiation of the swarmer cell into a stalked cell, full length PodJ is synthesized and localizes to the pole opposite the stalk. In late predivisional cells, full length PodJ is processed into a short form which remains localized at the flagellar pole after cell division and is degraded during swarmer to stalked cell differentiation. Polar localization of the developmental regulator PleC requires the presence of PodJ. In contrast, the polar localization of PodJ is not dependent on the presence of PleC. These results indicate that PodJ is an important determinant for the localization of a major regulator of cell differentiation. Thus, PodJ acts directly or indirectly to target PleC to the incipient swarmer pole, to establish the cellular asymmetry that leads to the synthesis of holdfasts and pili at their proper subcellular location.     

The asymmetric distribution of the essential histidine kinase PdhS indicates a differentiation event in Brucella abortus

Many organisms use polar localization of signalling proteins to control developmental events in response to completion of asymmetric cell division. Asymmetric division was recently reported for Brucella abortus, a class III facultative intracellular pathogen generating two sibling cells of slightly different size. Here we characterize PdhS, a cytoplasmic histidine kinase essential for B. abortus viability and homologous to the asymmetrically distributed PleC and DivJ histidine kinases from Caulobacter crescentus. PdhS is localized at the old pole of the large cell, and after division and growth, the small cell acquires PdhS at its old pole. PdhS may therefore be considered as a differentiation marker as it labels the old pole of the large cell. Moreover, PdhS colocalizes with its paired response regulator DivK. Finally, PdhS is able to localize at one pole in other alpha-proteobacteria, suggesting that a polar structure associating PdhS with one pole is conserved in these bacteria. We propose that a differentiation event takes place after the completion of cytokinesis in asymmetrically dividing alpha-proteobacteria. Altogether, these data suggest that prokaryotic differentiation may be much more widespread than expected.     

Allosteric regulation of histidine kinases by their cognate response regulator determines cell fate

The two-component phosphorylation network is of critical importance for bacterial growth and physiology. Here, we address plasticity and interconnection of distinct signal transduction pathways within this network. In Caulobacter crescentus antagonistic activities of the PleC phosphatase and DivJ kinase localized at opposite cell poles control the phosphorylation state and subcellular localization of the cell fate determinator protein DivK. We show that DivK functions as an allosteric regulator that switches PleC from a phosphatase into an autokinase state and thereby mediates a cyclic di-GMP-dependent morphogenetic program. Through allosteric activation of the DivJ autokinase, DivK also stimulates its own phosphorylation and polar localization. These data suggest that DivK is the central effector of an integrated circuit that operates via spatially organized feedback loops to control asymmetry and cell fate determination in C. crescentus. Thus, single domain response regulators can facilitate crosstalk, feedback control, and long-range communication among members of the two-component network.     

Cell cycle-dependent degradation of a flagellar motor component requires a novel-type response regulator

The poles of each Caulobacter crescentus cell undergo morphological development as a function of the cell cycle. A single flagellum assembled at one pole during the asymmetric cell division is later ejected and replaced by a newly synthesized stalk when the motile swarmer progeny differentiates into a sessile stalked cell. The removal of the flagellum during the swarmer-to-stalked cell transition coincides with the degradation of the FliF flagellar anchor protein. We report here that the cell cycle-dependent turnover of FliF does not require the structural components of the flagellum itself, arguing that it is the initial event leading to the ejection of the flagellum. Analysis of a polar development mutant, pleD, revealed that the pleD gene was required for efficient removal of FliF and for ejection of the flagellar structure during the swarmer-to-stalked cell transition. The PleD requirement for FliF degradation was also not dependent on the presence of any part of the flagellar structure. In addition, only 25% of the cells were able to synthesize a stalk during cell differentiation when PleD was absent. The pleD gene codes for a member of the response regulator family with a novel C-terminal regulatory domain. Mutational analysis confirmed that a highly conserved motif in the PleD C-terminal domain is essential to promote both FliF degradation and stalk biogenesis during cell differentiation. Signalling through the C-terminal domain of PleD is thus required for C. crescentus polar development. A second gene, fliL, was shown to be required for efficient turnover of FliF, but not for stalk biogenesis. The possible roles of PleD and FliL in C. crescentus polar development are discussed.     

Dissection of functional domains of the polar localization factor PodJ in Caulobacter crescentus

The polar organelle development protein, PodJ, is important for proper establishment of polarity in Caulobacter crescentus. podJ null mutants are unable to form holdfast or pili, have reduced swarming motility, and have difficulty ejecting the flagellum during the swarmer to stalked cell transition. In this study, we create a series of truncation mutants to investigate functional domains of PodJ. We show that PodJ has a transmembrane domain between amino acids 600 and 670. We identify a periplasmic region important for pili production and a cytoplasmic region required for holdfast formation and swarming motility, and establish that PleC localization is not required for holdfast formation and motility in soft agar. Analysis of the mutants reveals that the last 54 amino acids of the protein negatively regulate processing of the full-length form of the protein, PodJ(L), to a shorter form, PodJ(S). Finally, we identify a cytoplasmic region of PodJ involved in targeting it to the flagellar pole, and a periplasmic region required for localization of PleC.     

A dynamically localized histidine kinase controls the asymmetric distribution of polar pili proteins

Each cell division in Caulobacter crescentus is asymmetric, yielding a swarmer cell with several polar pili and a non-piliated stalked cell. To identify factors contributing to the asymmetric biogenesis of polar pili, cytological studies of pilus assembly components were performed. We show here that the CpaC protein, which is thought to form the outer membrane pilus secretion channel, and its assembly factor, CpaE, are localized to the cell pole prior to the polymerization of the pilus filament. We demonstrate that the PleC histidine kinase, a two-component signal transduction protein shown previously to localize to the piliated cell pole before and during pilus assembly, controls the accumulation of the pilin subunit, PilA. Using an inactive form of PleC (PleCH610A) that lacks the catalytic histidine residue, we provide evidence that PleC activity is responsible for the asymmetric distribution of CpaE and itself to only one of the two cell poles. Thus, a polar signal transduction protein controls its own asymmetric location as well as that of a factor assembling a polar organelle.     

Potential Role of a Bistable Histidine Kinase Switch in the Asymmetric Division Cycle of Caulobacter crescentus

The free-living aquatic bacterium, Caulobacter crescentus, exhibits two different morphologies during its life cycle. The morphological change from swarmer cell to stalked cell is a result of changes of function of two bi-functional histidine kinases, PleC and CckA. Here, we describe a detailed molecular mechanism by which the function of PleC changes between phosphatase and kinase state. By mathematical modeling of our proposed molecular interactions, we derive conditions under which PleC, CckA and its response regulators exhibit bistable behavior, thus providing a scenario for robust switching between swarmer and stalked states. Our simulations are in reasonable agreement with in vitro and in vivo experimental observations of wild type and mutant phenotypes. According to our model, the kinase form of PleC is essential for the swarmer-to-stalked transition and to prevent premature development of the swarmer pole. Based on our results, we reconcile some published experimental observations and suggest novel mutants to test our predictions.