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Spatial regulation in Caulobacter crescentus
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Spatial regulation in Caulobacter crescentus Martin Thanbichler1,2 The proper positioning of regulatory proteins has a central role in the organization of both prokaryotic and eukaryotic cells. Important insights into the principles that underlie the spatial control of cellular processes have been gained from studies on the asymmetric bacterium C. crescentus. In this organism, the cell cycle state is monitored by a complex two-component signaling network that feeds into the central pathways controlling gene expression, DNA replication and polar morphogenesis. Moreover, a sophisticated regulatory system closely interconnects chromosome dynamics with cell division, thus ensuring the generation of viable offspring. Recent work has identified several new key players in this intricate machinery and considerably increased our knowledge on the communication between the different regulatory pathways involved. Addresses 1 Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Straße, D-35043 Marburg, Germany 2 Laboratory for Microbiology, Department of Biology, Philipps University, Karl-von-Frisch-Straße 8, D-35043 Marburg, Germany Corresponding author: Thanbichler, Martin (thanbichler@mpi-marburg. mpg.de)
Current Opinion in Microbiology 2009, 12:715–721 This review comes from a themed issue on Growth and development: prokaryotes Edited by Patrick Viollier Available online 23rd October 2009 1369-5274/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2009.09.013
Introduction In recent years, it has become increasingly obvious that, next to temporal changes in gene expression, the spatial positioning of proteins has a key role in orchestrating the architecture and development of both prokaryotic and eukaryotic cells [1,2,3]. One of the leading model systems to study spatial regulation in bacteria is Caulobacter crescentus, a Gram-negative organism that is characterized by its asymmetric cell division [4–6]. C. crescentus starts its life cycle as a motile swarmer cell, carrying a cluster of type IV pili and a single polar flagellum at one of the cell poles (Figure 1A). After a mandatory differentiation process, which involves shedding of the flagellum, retraction of the pili, and establishment of an adhesive stalk at the previously flagellated pole, the cell starts to elongate and constrict. Later in the cell cycle, a new flagellum is assembled at the pole opposite the stalk. www.sciencedirect.com
Separation of the two siblings then yields a stalked cell, which immediately enters the next round of cell division, and a new swarmer cell, whose growth is again arrested until it transitions to the stalked cell stage. In C. crescentus, cell division is tightly synchronized with chromosome replication. The swarmer cell bears a single circular chromosome and rests in a replicationally quiescent state, reminiscent of the eukaryotic G1-phase. DNA replication initiates during the swarmer-to-stalked-cell transition and then proceeds throughout the rest of the division cycle, resulting in two daughter cells that each carry a single chromosome again. This review will discuss the most recent insights into the regulatory circuitry that determines the asymmetric organization of the C. crescentus cell and ensures tight coordination of morphogenesis and polar development with cell cycle progression.
Establishment of cellular asymmetry The C. crescentus cell cycle is driven by a regulatory cascade involving the sequential activation of four different master regulators [6]. One of these proteins, the twocomponent response regulator CtrA, is a key component of the circuitry that determines the differential fate of the two daughter cells [7]. In its phosphorylated state, CtrA activates or represses the transcription of more than 95 genes, responsible, for example, for DNA methylation, cell division, and flagellar biogenesis [8]. In addition, it binds to five sites within the chromosomal origin of replication, thereby blocking transition into S-phase [9,10,11]. CtrA is synthesized in a cell-cycle-dependent manner and starts to accumulate in the pre-divisional cell (Figure 1A). In addition, its activity is controlled at the post-translational level by both phosphorylation and proteolysis [7,12]. Formation of active CtrAP is only observed at the swarmer and pre-divisional stage, whereas it ceases upon entry into S-phase. Concurrent with its dephosphorylation, CtrA is targeted to the stalked pole [13] and degraded by the polarly localized ClpXP protease complex [14,15]. Thus, two complementary pathways promote removal of active CtrA at the beginning of a new replication cycle, thereby ensuring robust control of origin firing. Recent work has revealed the long-sought mechanism that underlies the control of CtrA activity (Figure 1B). Building on previous findings, phosphotransfer profiling has identified a signaling cascade that is responsible for the phosphorylation of CtrA, comprising the histidine kinase CckA and the histidine phosphotransferase ChpT [16]. In parallel, the same pathway was found to phosphorylate and thereby inactivate the single-domain Current Opinion in Microbiology 2009, 12:715–721
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Figure 1
Control of cell cycle progression and cellular asymmetry in C. crescentus. (A) Function of the PleC–DivJ–DivK signaling network. The phosphorylation state of the response regulator DivK is controlled both by DivJ, a histidine kinase, and PleC, a bifunctional enzyme that has either phosphatase (P) or histidine kinase (K) activity. While non-phosphorylated DivK is evenly distributed within the cell, its phosphorylated form interacts with the cell poles, leading to additional stimulation of the DivJ kinase activity and to a switch of PleC from the phosphatase to the kinase mode. Swarmer cells only contain PleC, working in the phosphatase mode, and thus accumulate non-phosphorylated DivK, which triggers a signaling cascade promoting the accumulation of CtrAP (green background color). After the swarmer-to-stalked cell transition, clusters of both PleC and DivJ are present in the cell, with PleC working in the kinase mode. As a consequence, DivK is largely converted into its phosphorylated form, thereby inducing elimination of CtrAP. Unlike DivK, the response regulator PleD is not a substrate of DivJ and specifically requires the kinase activity of PleC for its phosphorylation and polar localization. (B) Signaling pathways that tie cell cycle progression to CtrA phosphorylation and proteolysis. See text for further information.
response regulator CpdR, a factor required for polar localization of ClpXP and, thus, targeted CtrA degradation [16,17,18]. This wiring allows the cell to coordinately control both the activity and stability of CtrA in response to a shared input signal. The precise nature of the signal sensed by CckA is still unclear. However, its kinase activity is known to depend, presumably indirectly, on the phosphorylation state of another single-domain response regulator, named DivK, which Current Opinion in Microbiology 2009, 12:715–721
is part of a central checkpoint that monitors the cell cycle state of C. crescentus [16,19–21]. The activity of DivK is regulated by its dynamic interplay with the histidine kinase DivJ and the bifunctional histidine kinase/phosphatase PleC [19,21,22] (Figure 1A). At the swarmer cell stage, DivJ is hardly detectable, while PleC forms a complex at the flagellated pole, acting as a phosphatase on DivK [22,23]. Dephosphorylated DivK www.sciencedirect.com
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triggers the CckA-dependent phosphorelay and promotes the accumulation of CtrAP [16], thereby blocking initiation of chromosome replication. During transition to the stalked phase, the polar PleC cluster is gradually replaced by the DivJ kinase [23,24], leading to an increase in the level of DivKP [19,25]. Upon phosphorylation, DivK is able to interact with the future stalked pole [22,25]. Recent findings indicate that the consequent increase in the local concentration of DivK results in additional stimulation of DivJ activity and in transition of PleC from the phosphatase to the kinase mode [24]. This positive feedback loop might ensure rapid and robust phosphorylation of DivK in the stalked cell, thereby promoting the elimination of CtrAP and entry into S-phase. Later in the cell cycle, PleC assembles into a new cluster at the pole opposite the stalk [23], which colocalizes with of a second, newly formed DivKP cluster [22,25], thus still functioning in the kinase mode [24]. Once cytokinesis compartmentalizes the two daughter cells, the DivK pool is divided into two different populations [21]. The molecules captured in the stalked sibling are left with DivJ and, therefore, retained in the phosphorylated state [22], allowing a new round of replication to initiate. Those segregated into the swarmer sibling, by contrast, are isolated from their major kinase DivJ and only able to interact with PleC. Owing to the lower phosphorylation rates and, possibly, residual PleC phosphatase activity, the levels of DivKP start to decrease, promoting disassembly of the polar DivK complex [22,25] and thus reversion of PleC from the kinase to the phosphatase mode [24]. After this switch in PleC activity, the DivK molecules contained in the swarmer cell are largely converted to their non-phosphorylated state [25], resulting in the re-accumulation of CtrAP and in an arrest of the cell in G1-phase. Apart from its pivotal role in cell differentiation, the PleC–DivJ–DivK signaling network controls various processes involved in the adaptation of C. crescentus to a sessile lifestyle, including flagellar ejection, stalk biogenesis, and surface attachment [26,27]. This regulatory effect is mediated by PleD, an unusual response regulator that carries a C-terminal guanylate cyclase domain [28,29]. During the swarmer-to-stalked-cell transition, PleD is phosphorylated by the kinase activity of PleC [24] and thus stimulated to dimerize [30] (Figure 1B). Its dimeric form is then recruited to the flagellated pole and induced to synthesize the second messenger c-diGMP [30,31], a signaling molecule promoting transition from motility to sessility in a wide range of bacteria [32]. Both the polar localization of PleD and the (possibly local) action of the second messenger are subsequently responsible for the establishment of a mature stalked pole, able to promote surface attachment. Interestingly, the adhesive properties of C. crescentus were found to be additionally modulated by light, mediated through the LovRK two-component system [33]. However, the www.sciencedirect.com
targets of this regulatory pathway and its biological significance are still unknown. C-di-GMP was shown to have a second key function in C. crescentus, acting as a co-inducer of CtrA degradation. The protein mediating this activity is PopA, a structural homologue of PleD that contains a catalytically inactive guanylate cyclase domain serving as a c-di-GMP sensor [34]. During the swarmer-to-stalked-cell transition, PopA is sequestered to the future stalked pole in a ligand-dependent manner, promoting recruitment of CtrA to the polar ClpXP protease complex via direct interaction with the proteolytic regulator RcdA. However, given that C. crescentus contains a variety of proteins implicated in c-di-GMP metabolism, the mechanism that times the accumulation of this signaling molecule and, thus, PopA activity is still obscure. A crucial parameter in the function of DivK is the proper spatial arrangement of its interaction partners PleC and DivJ. Localization of PleC to the newly generated flagellated pole is dependent on PodJ, a coiled-coil protein also required for polar pilus biogenesis [35,36]. Recent work has now identified the determinant responsible for recruitment of DivJ to the stalked cell pole [37]. This novel factor, termed SpmX, is essential for both the positioning and activity of DivJ, thus lying at the heart of the circuitry that regulates the C. crescentus cell cycle. However, what are the upstream factors that, in turn, ensure proper positioning of PodJ and SpmX? While the situation is unclear for PodJ, SpmX was found to contain a lysozyme-like domain that is necessary and sufficient for its localization [37]. It might, therefore, recognize structural cues in the cell wall, possibly originating from the establishment of the stalk structure.
Regulation of chromosome dynamics and cell division In C. crescentus, the regulatory circuits controlling cell cycle progression and chromosome dynamics are tightly interwoven. Once the PleC–DivJ–DivK signaling network and the activation of PopA by c-di-GMP trigger clearance of CtrAP from the cell, the replication initiator protein DnaA gains access to the chromosomal origin of replication, inducing strand separation and replisome assembly [10,38]. The two replication forks then move bidirectionally along the left and right arm of the chromosome until they meet in the terminus region. Given that CtrA levels remain low throughout S-phase, supernumerary initiation events must be suppressed by other, CtrA-independent mechanisms. Consistently, recent work has shown that inactivation of DnaA by the replisome-associated protein Hda plays an important role in the attenuation of origin firing during later stages of the cell cycle [39]. Duplication of the chromosome induces significant rearrangements within the nucleoid. In swarmer cells, the Current Opinion in Microbiology 2009, 12:715–721
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replication origin is located at the flagellated pole [40], while all other loci are arranged within the cell in a linear order according to their position on the circular chromosomal map [41]. Upon entry into S-phase, the two copies of the origin region are rapidly separated from each other and positioned at opposite cell poles [41], serving as the corner stones of the two nascent sister nucleoids. Subsequent stretches of newly synthesized DNA are successively moved into the daughter cell compartments as replication is in progress [41]. They are re-compacted and stacked upon each other in the form of supercoiled loops [42], thus maintaining the original ordering of loci. Concomitantly, the replisome moves slowly from its initial, polar position towards the cell center [43], where duplication of the terminus region takes place [41]. Whereas the bulk of chromosomal DNA may be partitioned by entropic effects and the forces resulting from DNA re-condensation, origin movement is probably driven by a dedicated segregation apparatus, involving the ParAB chromosome partitioning system [44,45,46,47]. ParB is a DNA-binding protein that associates with a cluster of specific sites ( parS) in the vicinity of the chromosomal replication origin [47]. After initial attachment, it is thought to spread into the flanking regions, assembling into a large nucleoprotein complex that covers several kilobases of DNA. This polymeric structure interacts with the Walker ATPase ParA, which has been postulated to constitute a mitotic-like apparatus responsible for origin segregation [48,49]. A recent study indeed showed that plasmid-borne copies of parS interfere with proper chromosome partitioning [45]. Moreover, the parS cluster was found to be among the first chromosomal loci to be segregated, even when transplanted to an ectopic site distal from the origin of replication [45]. Along with the finding that changes in the levels of ParA or ParB [46,47] as well as impairment of the ParA ATPase activity [45] result in severe chromosome segregation defects, these results strongly support a role for the ParAB system in origin movement. Interestingly, parS sites also emerged to mediate polar attachment of the origin regions [45]. The underlying mechanism was clarified by the identification of a polymeric protein, PopZ, that lines the cell poles and tightly interacts with ParB, serving as an anchor for the ParB–parS nucleoprotein complex [50,51] (Figure 2). PopZ shows a dynamic localization pattern similar to that of the origin regions, with G1-phase cells bearing a single complex at their old pole. Upon initiation of chromosome replication, a second complex is established at the opposite end of the cell, ready to capture the moving origin copy once it arrives at its new destination [50,51]. The factors triggering this change in localization are largely unknown, even though PopZ assembly was proposed to occur preferentially in cellular regions devoid of chromosomal DNA [51]. Aside from its role in origin attachment, PopZ was found to interact with the histidine kinases CckA and DivJ Current Opinion in Microbiology 2009, 12:715–721
Figure 2
Coordination of chromosome dynamics and cell division. G1-phase cells contain a single chromosome, whose origin is tethered to the old pole through association of the anchoring protein PopZ with the ParB–parS nucleoprotein complex. The cell division regulator MipZ interacts with ParB, forming a linear gradient that restricts polymerization of FtsZ to the new pole. Upon entry into S-phase, the origin region is duplicated and its copies are rapidly segregated from each other. While one of them remains at the original location, the other one traverses the cell and assumes a position at the opposite end of the cell, where it is captured by a second, newly formed PopZ cluster. The concurrent recruitment of MipZ to both cell poles leads to disintegration of the polar FtsZ complex and assembly of an FtsZ ring in proximity of the cell center, the region of lowest MipZ concentration.
[51], suggesting that it could serve as an integration platform for processes involved in cell polarization. Polar attachment of the ParB–parS nucleoprotein complex not only contributes to chromosome partitioning, but it also sets the basis for proper cell division (Figure 2). Formation of the division apparatus requires polymerization of the tubulin homologue FtsZ into a polymeric www.sciencedirect.com
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annular structure, called the Z-ring [52]. In C. crescentus, this assembly process is negatively regulated by the Walker ATPase MipZ, a homologue of ParA that associates with ParB and, therefore, closely follows the movement of the origin regions [53]. As a consequence, MipZ is concentrated at the old pole in G1-phase, whereas it assumes a bipolar localization pattern once replication initiates and the two origin copies are segregated to opposite ends of the cell. Owing to the highly dynamic nature of the MipZ–ParB complex, MipZ is not stably tethered to the origin regions but rather distributed in a gradient, with its concentration being highest at the cell poles and lowest at midcell. Given that MipZ acts as an inhibitor of FtsZ polymerization, this skewed distribution ensures that assembly of the division apparatus can only occur close to the cell center, in-between the two nascent sister nucleoids [53]. Apart from separating the two daughter cell compartments, the cell division apparatus plays a vital role in chromosome segregation, cell polarity, and morphogenesis. One of its components, FtsK, serves as a DNA translocase that sorts the newly replicated chromosomes into the two daughter cell compartments. Moreover, FtsK is crucial for chromosome decatenation and in the resolution of chromosome dimers, two processes that are required to finalize the segregation process [54,55]. Another constituent of the cell division apparatus is TipN, a coiled-coil protein that is retained at the previous cell division site, acting as a birthmark that labels the new cell pole and thus ensures proper positioning of the flagellum during the subsequent cell cycle [56,57]. TipN exerts its regulatory effect by mediating the polar localization of another protein, TipF, which is required for the initiation of flagellar assembly [56]. Interestingly, prolonged overproduction of TipN induces the formation of ectopic cell poles, suggesting that the protein could have a global function in cell polarization [57]. In addition to TipN, the protein PflI was found to contribute to the robustness of flagellar placement [58]. However, the mechanism of its localization and action has so far remained unknown. Finally, recent work showed that the cell division apparatus not only mediates constriction but also elongation of the C. crescentus cell [59,60], thus helping establish the coordinate system that guides the positioning and function of other spatial regulators. This observation is consistent with the fact that MurG, the enzyme catalyzing the last step of peptidoglycan precursor synthesis, is recruited to midcell in an FtsZ-dependent manner. There it might cooperate with septal cell wall synthases in extending the peptidoglycan sacculus [59,60], independent of the generic MreB- and RodZ-dependent morphogenetic system [61].
Conclusions Recent advances have significantly increased our knowledge of the processes that spatially organize the C. crescentus www.sciencedirect.com
cell. The studies discussed above have identified various long-sought missing links in the two-component signaling network that controls the activity of CtrA and other key regulatory proteins. Moreover, they have provided intriguing insights into the mechanisms that regulate and coordinate cell polarization, chromosome dynamics, and cell division. Nevertheless, a number of questions remain to be answered. Importantly, the pathway that ties CckA activity to the phosphorylation state of DivK is still unclear. Other open issues are the control of c-di-GMP metabolism and the precise function of the machinery that targets CtrA to the polar ClpXP protease. Given the multiple functions of ParB, it will also be interesting to determine if the ParB– parS complex serves as a communication platform that allows regulatory crosstalk between the cellular processes it mediates. Finally, it will be crucial to determine the cues that direct localized proteins to their subcellular destinations. Addressing these and many other problems might eventually allow us to completely describe the regulatory network that controls the temporal and spatial organization of C. crescentus and, thus, to understand the basic principles that constitute an asymmetric bacterial cell.
Acknowledgements This work was supported by the Max Planck Society and a Young Investigator Grant from the Human Frontier Science Program (HFSP).
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55. Bigot S, Sivanathan V, Possoz C, Barre FX, Cornet F: FtsK, a literate chromosome segregation machine. Mol Microbiol 2007, 64:1434-1441.
45. Toro E, Hong SH, McAdams HH, Shapiro L: Caulobacter requires a dedicated mechanism to initiate chromosome segregation. Proc Natl Acad Sci U S A 2008, 105:15435-15440. This paper provides convincing evidence for the long-standing hypothesis that segregation of the origin regions is largely mediated by the ParAB DNA-partitioning system in C. crescentus. The binding sites for the centromer-binding protein ParB ( parS) are shown to impair the segregation process when introduced in trans. Moreover, using defined chromosome inversions and site-specific fluorescence labeling, the parS sites are identified as attachment points for the machineries mediating segregation and polar anchoring of the origin regions.
56. Huitema E, Pritchard S, Matteson D, Radhakrishnan SK, Viollier PH: Bacterial birth scar proteins mark future flagellum assembly site. Cell 2006, 124:1025-1037.
46. Mohl DA, Easter J Jr, Gober JW: The chromosome partitioning protein, ParB, is required for cytokinesis in Caulobacter crescentus. Mol Microbiol 2001, 42:741-755.
59. Aaron M, Charbon G, Lam H, Schwarz H, Vollmer W, Jacobs Wagner C: The tubulin homologue FtsZ contributes to cell elongation by guiding cell wall precursor synthesis in Caulobacter crescentus. Mol Microbiol 2007, 64:938-952. This study demonstrates that C. crescentus shows FtsZ-dependent medial growth, occurring independently of the MreB cytoskeleton. This observation is correlated with the recruitment of MurG, an enzyme involved in the synthesis of peptidoglycan precursors, to the cell division site.
47. Mohl DA, Gober JW: Cell cycle-dependent polar localization of chromosome partitioning proteins in Caulobacter crescentus. Cell 1997, 88:675-684. 48. Figge RM, Easter J, Gober JW: Productive interaction between the chromosome partitioning proteins, ParA and ParB, is required for the progression of the cell cycle in Caulobacter crescentus. Mol Microbiol 2003, 47:1225-1237. 49. Easter J Jr, Gober JW: ParB-stimulated nucleotide exchange regulates a switch in functionally distinct ParA activities. Mol Cell 2002, 10:427-434. 50. Bowman GR, Comolli LR, Zhu J, Eckart M, Koenig M, Downing KH, Moerner WE, Earnest T, Shapiro L: A polymeric protein anchors the chromosomal origin/ParB complex at a bacterial cell pole. Cell 2008, 134:945-955. (see comment on ref. [51]). 51. Ebersbach G, Briegel A, Jensen GJ, Jacobs-Wagner C: A self associating protein critical for chromosome attachment,
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57. Lam H, Schofield WB, Jacobs-Wagner C: A landmark protein essential for establishing and perpetuating the polarity of a bacterial cell. Cell 2006, 124:1011-1023. 58. Obuchowski PL, Jacobs-Wagner C: PflI, a protein involved in flagellar positioning in Caulobacter crescentus. J Bacteriol 2008, 190:1718-1729.
60. Divakaruni AV, Baida C, White CL, Gober JW: The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes. Mol Microbiol 2007, 66:174-188. Similar to the work of Aaron et al. [59], this paper shows the existence of a medial growth zone in C. crescentus. 61. Alyahya SA, Alexander R, Costa T, Henriques AO, Emonet T, Jacobs-Wagner C: RodZ, a component of the bacterial core morphogenic apparatus. Proc Natl Acad Sci U S A 2009, 106:1239-1244. This study reports the identification of a highly conserved membrane protein that mediates interaction between the MreB cytoskeleton and the cell wall biosynthetic machinery.
Current Opinion in Microbiology 2009, 12:715–721
Spatial regulation in Caulobacter crescentus Martin Thanbichler1,2 The proper positioning of regulatory proteins has a central role in the organization of both prokaryotic and eukaryotic cells. Important insights into the principles that underlie the spatial control of cellular processes have been gained from studies on the asymmetric bacterium C. crescentus. In this organism, the cell cycle state is monitored by a complex two-component signaling network that feeds into the central pathways controlling gene expression, DNA replication and polar morphogenesis. Moreover, a sophisticated regulatory system closely interconnects chromosome dynamics with cell division, thus ensuring the generation of viable offspring. Recent work has identified several new key players in this intricate machinery and considerably increased our knowledge on the communication between the different regulatory pathways involved. Addresses 1 Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Straße, D-35043 Marburg, Germany 2 Laboratory for Microbiology, Department of Biology, Philipps University, Karl-von-Frisch-Straße 8, D-35043 Marburg, Germany Corresponding author: Thanbichler, Martin (thanbichler@mpi-marburg. mpg.de)
Current Opinion in Microbiology 2009, 12:715–721 This review comes from a themed issue on Growth and development: prokaryotes Edited by Patrick Viollier Available online 23rd October 2009 1369-5274/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2009.09.013
Introduction In recent years, it has become increasingly obvious that, next to temporal changes in gene expression, the spatial positioning of proteins has a key role in orchestrating the architecture and development of both prokaryotic and eukaryotic cells [1,2,3]. One of the leading model systems to study spatial regulation in bacteria is Caulobacter crescentus, a Gram-negative organism that is characterized by its asymmetric cell division [4–6]. C. crescentus starts its life cycle as a motile swarmer cell, carrying a cluster of type IV pili and a single polar flagellum at one of the cell poles (Figure 1A). After a mandatory differentiation process, which involves shedding of the flagellum, retraction of the pili, and establishment of an adhesive stalk at the previously flagellated pole, the cell starts to elongate and constrict. Later in the cell cycle, a new flagellum is assembled at the pole opposite the stalk. www.sciencedirect.com
Separation of the two siblings then yields a stalked cell, which immediately enters the next round of cell division, and a new swarmer cell, whose growth is again arrested until it transitions to the stalked cell stage. In C. crescentus, cell division is tightly synchronized with chromosome replication. The swarmer cell bears a single circular chromosome and rests in a replicationally quiescent state, reminiscent of the eukaryotic G1-phase. DNA replication initiates during the swarmer-to-stalked-cell transition and then proceeds throughout the rest of the division cycle, resulting in two daughter cells that each carry a single chromosome again. This review will discuss the most recent insights into the regulatory circuitry that determines the asymmetric organization of the C. crescentus cell and ensures tight coordination of morphogenesis and polar development with cell cycle progression.
Establishment of cellular asymmetry The C. crescentus cell cycle is driven by a regulatory cascade involving the sequential activation of four different master regulators [6]. One of these proteins, the twocomponent response regulator CtrA, is a key component of the circuitry that determines the differential fate of the two daughter cells [7]. In its phosphorylated state, CtrA activates or represses the transcription of more than 95 genes, responsible, for example, for DNA methylation, cell division, and flagellar biogenesis [8]. In addition, it binds to five sites within the chromosomal origin of replication, thereby blocking transition into S-phase [9,10,11]. CtrA is synthesized in a cell-cycle-dependent manner and starts to accumulate in the pre-divisional cell (Figure 1A). In addition, its activity is controlled at the post-translational level by both phosphorylation and proteolysis [7,12]. Formation of active CtrAP is only observed at the swarmer and pre-divisional stage, whereas it ceases upon entry into S-phase. Concurrent with its dephosphorylation, CtrA is targeted to the stalked pole [13] and degraded by the polarly localized ClpXP protease complex [14,15]. Thus, two complementary pathways promote removal of active CtrA at the beginning of a new replication cycle, thereby ensuring robust control of origin firing. Recent work has revealed the long-sought mechanism that underlies the control of CtrA activity (Figure 1B). Building on previous findings, phosphotransfer profiling has identified a signaling cascade that is responsible for the phosphorylation of CtrA, comprising the histidine kinase CckA and the histidine phosphotransferase ChpT [16]. In parallel, the same pathway was found to phosphorylate and thereby inactivate the single-domain Current Opinion in Microbiology 2009, 12:715–721
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Figure 1
Control of cell cycle progression and cellular asymmetry in C. crescentus. (A) Function of the PleC–DivJ–DivK signaling network. The phosphorylation state of the response regulator DivK is controlled both by DivJ, a histidine kinase, and PleC, a bifunctional enzyme that has either phosphatase (P) or histidine kinase (K) activity. While non-phosphorylated DivK is evenly distributed within the cell, its phosphorylated form interacts with the cell poles, leading to additional stimulation of the DivJ kinase activity and to a switch of PleC from the phosphatase to the kinase mode. Swarmer cells only contain PleC, working in the phosphatase mode, and thus accumulate non-phosphorylated DivK, which triggers a signaling cascade promoting the accumulation of CtrAP (green background color). After the swarmer-to-stalked cell transition, clusters of both PleC and DivJ are present in the cell, with PleC working in the kinase mode. As a consequence, DivK is largely converted into its phosphorylated form, thereby inducing elimination of CtrAP. Unlike DivK, the response regulator PleD is not a substrate of DivJ and specifically requires the kinase activity of PleC for its phosphorylation and polar localization. (B) Signaling pathways that tie cell cycle progression to CtrA phosphorylation and proteolysis. See text for further information.
response regulator CpdR, a factor required for polar localization of ClpXP and, thus, targeted CtrA degradation [16,17,18]. This wiring allows the cell to coordinately control both the activity and stability of CtrA in response to a shared input signal. The precise nature of the signal sensed by CckA is still unclear. However, its kinase activity is known to depend, presumably indirectly, on the phosphorylation state of another single-domain response regulator, named DivK, which Current Opinion in Microbiology 2009, 12:715–721
is part of a central checkpoint that monitors the cell cycle state of C. crescentus [16,19–21]. The activity of DivK is regulated by its dynamic interplay with the histidine kinase DivJ and the bifunctional histidine kinase/phosphatase PleC [19,21,22] (Figure 1A). At the swarmer cell stage, DivJ is hardly detectable, while PleC forms a complex at the flagellated pole, acting as a phosphatase on DivK [22,23]. Dephosphorylated DivK www.sciencedirect.com
Spatial regulation in Caulobacter crescentus Thanbichler 717
triggers the CckA-dependent phosphorelay and promotes the accumulation of CtrAP [16], thereby blocking initiation of chromosome replication. During transition to the stalked phase, the polar PleC cluster is gradually replaced by the DivJ kinase [23,24], leading to an increase in the level of DivKP [19,25]. Upon phosphorylation, DivK is able to interact with the future stalked pole [22,25]. Recent findings indicate that the consequent increase in the local concentration of DivK results in additional stimulation of DivJ activity and in transition of PleC from the phosphatase to the kinase mode [24]. This positive feedback loop might ensure rapid and robust phosphorylation of DivK in the stalked cell, thereby promoting the elimination of CtrAP and entry into S-phase. Later in the cell cycle, PleC assembles into a new cluster at the pole opposite the stalk [23], which colocalizes with of a second, newly formed DivKP cluster [22,25], thus still functioning in the kinase mode [24]. Once cytokinesis compartmentalizes the two daughter cells, the DivK pool is divided into two different populations [21]. The molecules captured in the stalked sibling are left with DivJ and, therefore, retained in the phosphorylated state [22], allowing a new round of replication to initiate. Those segregated into the swarmer sibling, by contrast, are isolated from their major kinase DivJ and only able to interact with PleC. Owing to the lower phosphorylation rates and, possibly, residual PleC phosphatase activity, the levels of DivKP start to decrease, promoting disassembly of the polar DivK complex [22,25] and thus reversion of PleC from the kinase to the phosphatase mode [24]. After this switch in PleC activity, the DivK molecules contained in the swarmer cell are largely converted to their non-phosphorylated state [25], resulting in the re-accumulation of CtrAP and in an arrest of the cell in G1-phase. Apart from its pivotal role in cell differentiation, the PleC–DivJ–DivK signaling network controls various processes involved in the adaptation of C. crescentus to a sessile lifestyle, including flagellar ejection, stalk biogenesis, and surface attachment [26,27]. This regulatory effect is mediated by PleD, an unusual response regulator that carries a C-terminal guanylate cyclase domain [28,29]. During the swarmer-to-stalked-cell transition, PleD is phosphorylated by the kinase activity of PleC [24] and thus stimulated to dimerize [30] (Figure 1B). Its dimeric form is then recruited to the flagellated pole and induced to synthesize the second messenger c-diGMP [30,31], a signaling molecule promoting transition from motility to sessility in a wide range of bacteria [32]. Both the polar localization of PleD and the (possibly local) action of the second messenger are subsequently responsible for the establishment of a mature stalked pole, able to promote surface attachment. Interestingly, the adhesive properties of C. crescentus were found to be additionally modulated by light, mediated through the LovRK two-component system [33]. However, the www.sciencedirect.com
targets of this regulatory pathway and its biological significance are still unknown. C-di-GMP was shown to have a second key function in C. crescentus, acting as a co-inducer of CtrA degradation. The protein mediating this activity is PopA, a structural homologue of PleD that contains a catalytically inactive guanylate cyclase domain serving as a c-di-GMP sensor [34]. During the swarmer-to-stalked-cell transition, PopA is sequestered to the future stalked pole in a ligand-dependent manner, promoting recruitment of CtrA to the polar ClpXP protease complex via direct interaction with the proteolytic regulator RcdA. However, given that C. crescentus contains a variety of proteins implicated in c-di-GMP metabolism, the mechanism that times the accumulation of this signaling molecule and, thus, PopA activity is still obscure. A crucial parameter in the function of DivK is the proper spatial arrangement of its interaction partners PleC and DivJ. Localization of PleC to the newly generated flagellated pole is dependent on PodJ, a coiled-coil protein also required for polar pilus biogenesis [35,36]. Recent work has now identified the determinant responsible for recruitment of DivJ to the stalked cell pole [37]. This novel factor, termed SpmX, is essential for both the positioning and activity of DivJ, thus lying at the heart of the circuitry that regulates the C. crescentus cell cycle. However, what are the upstream factors that, in turn, ensure proper positioning of PodJ and SpmX? While the situation is unclear for PodJ, SpmX was found to contain a lysozyme-like domain that is necessary and sufficient for its localization [37]. It might, therefore, recognize structural cues in the cell wall, possibly originating from the establishment of the stalk structure.
Regulation of chromosome dynamics and cell division In C. crescentus, the regulatory circuits controlling cell cycle progression and chromosome dynamics are tightly interwoven. Once the PleC–DivJ–DivK signaling network and the activation of PopA by c-di-GMP trigger clearance of CtrAP from the cell, the replication initiator protein DnaA gains access to the chromosomal origin of replication, inducing strand separation and replisome assembly [10,38]. The two replication forks then move bidirectionally along the left and right arm of the chromosome until they meet in the terminus region. Given that CtrA levels remain low throughout S-phase, supernumerary initiation events must be suppressed by other, CtrA-independent mechanisms. Consistently, recent work has shown that inactivation of DnaA by the replisome-associated protein Hda plays an important role in the attenuation of origin firing during later stages of the cell cycle [39]. Duplication of the chromosome induces significant rearrangements within the nucleoid. In swarmer cells, the Current Opinion in Microbiology 2009, 12:715–721
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replication origin is located at the flagellated pole [40], while all other loci are arranged within the cell in a linear order according to their position on the circular chromosomal map [41]. Upon entry into S-phase, the two copies of the origin region are rapidly separated from each other and positioned at opposite cell poles [41], serving as the corner stones of the two nascent sister nucleoids. Subsequent stretches of newly synthesized DNA are successively moved into the daughter cell compartments as replication is in progress [41]. They are re-compacted and stacked upon each other in the form of supercoiled loops [42], thus maintaining the original ordering of loci. Concomitantly, the replisome moves slowly from its initial, polar position towards the cell center [43], where duplication of the terminus region takes place [41]. Whereas the bulk of chromosomal DNA may be partitioned by entropic effects and the forces resulting from DNA re-condensation, origin movement is probably driven by a dedicated segregation apparatus, involving the ParAB chromosome partitioning system [44,45,46,47]. ParB is a DNA-binding protein that associates with a cluster of specific sites ( parS) in the vicinity of the chromosomal replication origin [47]. After initial attachment, it is thought to spread into the flanking regions, assembling into a large nucleoprotein complex that covers several kilobases of DNA. This polymeric structure interacts with the Walker ATPase ParA, which has been postulated to constitute a mitotic-like apparatus responsible for origin segregation [48,49]. A recent study indeed showed that plasmid-borne copies of parS interfere with proper chromosome partitioning [45]. Moreover, the parS cluster was found to be among the first chromosomal loci to be segregated, even when transplanted to an ectopic site distal from the origin of replication [45]. Along with the finding that changes in the levels of ParA or ParB [46,47] as well as impairment of the ParA ATPase activity [45] result in severe chromosome segregation defects, these results strongly support a role for the ParAB system in origin movement. Interestingly, parS sites also emerged to mediate polar attachment of the origin regions [45]. The underlying mechanism was clarified by the identification of a polymeric protein, PopZ, that lines the cell poles and tightly interacts with ParB, serving as an anchor for the ParB–parS nucleoprotein complex [50,51] (Figure 2). PopZ shows a dynamic localization pattern similar to that of the origin regions, with G1-phase cells bearing a single complex at their old pole. Upon initiation of chromosome replication, a second complex is established at the opposite end of the cell, ready to capture the moving origin copy once it arrives at its new destination [50,51]. The factors triggering this change in localization are largely unknown, even though PopZ assembly was proposed to occur preferentially in cellular regions devoid of chromosomal DNA [51]. Aside from its role in origin attachment, PopZ was found to interact with the histidine kinases CckA and DivJ Current Opinion in Microbiology 2009, 12:715–721
Figure 2
Coordination of chromosome dynamics and cell division. G1-phase cells contain a single chromosome, whose origin is tethered to the old pole through association of the anchoring protein PopZ with the ParB–parS nucleoprotein complex. The cell division regulator MipZ interacts with ParB, forming a linear gradient that restricts polymerization of FtsZ to the new pole. Upon entry into S-phase, the origin region is duplicated and its copies are rapidly segregated from each other. While one of them remains at the original location, the other one traverses the cell and assumes a position at the opposite end of the cell, where it is captured by a second, newly formed PopZ cluster. The concurrent recruitment of MipZ to both cell poles leads to disintegration of the polar FtsZ complex and assembly of an FtsZ ring in proximity of the cell center, the region of lowest MipZ concentration.
[51], suggesting that it could serve as an integration platform for processes involved in cell polarization. Polar attachment of the ParB–parS nucleoprotein complex not only contributes to chromosome partitioning, but it also sets the basis for proper cell division (Figure 2). Formation of the division apparatus requires polymerization of the tubulin homologue FtsZ into a polymeric www.sciencedirect.com
Spatial regulation in Caulobacter crescentus Thanbichler 719
annular structure, called the Z-ring [52]. In C. crescentus, this assembly process is negatively regulated by the Walker ATPase MipZ, a homologue of ParA that associates with ParB and, therefore, closely follows the movement of the origin regions [53]. As a consequence, MipZ is concentrated at the old pole in G1-phase, whereas it assumes a bipolar localization pattern once replication initiates and the two origin copies are segregated to opposite ends of the cell. Owing to the highly dynamic nature of the MipZ–ParB complex, MipZ is not stably tethered to the origin regions but rather distributed in a gradient, with its concentration being highest at the cell poles and lowest at midcell. Given that MipZ acts as an inhibitor of FtsZ polymerization, this skewed distribution ensures that assembly of the division apparatus can only occur close to the cell center, in-between the two nascent sister nucleoids [53]. Apart from separating the two daughter cell compartments, the cell division apparatus plays a vital role in chromosome segregation, cell polarity, and morphogenesis. One of its components, FtsK, serves as a DNA translocase that sorts the newly replicated chromosomes into the two daughter cell compartments. Moreover, FtsK is crucial for chromosome decatenation and in the resolution of chromosome dimers, two processes that are required to finalize the segregation process [54,55]. Another constituent of the cell division apparatus is TipN, a coiled-coil protein that is retained at the previous cell division site, acting as a birthmark that labels the new cell pole and thus ensures proper positioning of the flagellum during the subsequent cell cycle [56,57]. TipN exerts its regulatory effect by mediating the polar localization of another protein, TipF, which is required for the initiation of flagellar assembly [56]. Interestingly, prolonged overproduction of TipN induces the formation of ectopic cell poles, suggesting that the protein could have a global function in cell polarization [57]. In addition to TipN, the protein PflI was found to contribute to the robustness of flagellar placement [58]. However, the mechanism of its localization and action has so far remained unknown. Finally, recent work showed that the cell division apparatus not only mediates constriction but also elongation of the C. crescentus cell [59,60], thus helping establish the coordinate system that guides the positioning and function of other spatial regulators. This observation is consistent with the fact that MurG, the enzyme catalyzing the last step of peptidoglycan precursor synthesis, is recruited to midcell in an FtsZ-dependent manner. There it might cooperate with septal cell wall synthases in extending the peptidoglycan sacculus [59,60], independent of the generic MreB- and RodZ-dependent morphogenetic system [61].
Conclusions Recent advances have significantly increased our knowledge of the processes that spatially organize the C. crescentus www.sciencedirect.com
cell. The studies discussed above have identified various long-sought missing links in the two-component signaling network that controls the activity of CtrA and other key regulatory proteins. Moreover, they have provided intriguing insights into the mechanisms that regulate and coordinate cell polarization, chromosome dynamics, and cell division. Nevertheless, a number of questions remain to be answered. Importantly, the pathway that ties CckA activity to the phosphorylation state of DivK is still unclear. Other open issues are the control of c-di-GMP metabolism and the precise function of the machinery that targets CtrA to the polar ClpXP protease. Given the multiple functions of ParB, it will also be interesting to determine if the ParB– parS complex serves as a communication platform that allows regulatory crosstalk between the cellular processes it mediates. Finally, it will be crucial to determine the cues that direct localized proteins to their subcellular destinations. Addressing these and many other problems might eventually allow us to completely describe the regulatory network that controls the temporal and spatial organization of C. crescentus and, thus, to understand the basic principles that constitute an asymmetric bacterial cell.
Acknowledgements This work was supported by the Max Planck Society and a Young Investigator Grant from the Human Frontier Science Program (HFSP).
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10. Bastedo DP, Marczynski GT: CtrA response regulator binding to the Caulobacter chromosome replication origin is required during nutrient and antibiotic stress as well as during cell cycle progression. Mol Microbiol 2009, 72:139-154. This study re-investigates the role of the cell cycle regulator CtrA in the regulation of replication initiation. CtrA is found to be required for suppressing origin firing in the swarmer cell but dispensable for preventing supernumerary rounds of replication after transition into S-phase. 11. Shaheen SM, Ouimet MC, Marczynski GT: Comparative analysis of Caulobacter chromosome replication origins. Microbiology 2009, 155:1215-1225. 12. Domian IJ, Quon KC, Shapiro L: Cell type-specific phosphorylation and proteolysis of a transcriptional regulator controls the G1-to-S transition in a bacterial cell cycle. Cell 1997, 90:415-424. 13. Ryan KR, Huntwork S, Shapiro L: Recruitment of a cytoplasmic response regulator to the cell pole is linked to its cell cycleregulated proteolysis. Proc Natl Acad Sci U S A 2004, 101:7415-7420. 14. Jenal U, Fuchs T: An essential protease involved in bacterial cell-cycle control. EMBO J 1998, 17:5658-5669. 15. McGrath PT, Iniesta AA, Ryan KR, Shapiro L, McAdams HH: A dynamically localized protease complex and a polar specificity factor control a cell cycle master regulator. Cell 2006, 124:535-547. 16. Biondi EG, Reisinger SJ, Skerker JM, Arif M, Perchuk BS, Ryan KR, Laub MT: Regulation of the bacterial cell cycle by an integrated genetic circuit. Nature 2006, 444:899-904. 17. Iniesta AA, McGrath PT, Reisenauer A, McAdams HH, Shapiro L: A phospho-signaling pathway controls the localization and activity of a protease complex critical for bacterial cell cycle progression. Proc Natl Acad Sci U S A 2006, 103:10935-10940. 18. Iniesta AA, Shapiro L: A bacterial control circuit integrates polar localization and proteolysis of key regulatory proteins with a phospho-signaling cascade. Proc Natl Acad Sci U S A 2008, 105:16602-16607. This paper reports a feedback loop that facilitates re-accumulation of the cell cycle regulator CtrA after its degradation by the ClpXP protease during the G1-to-S-transition. CpdR, a response regulator that is required for the activation of ClpXP is found to be itself subject to ClpXP-mediated proteolysis, thereby stimulating its own inactivation and, thus, effecting stabilization of CtrA. 19. Hecht GB, Lane T, Ohta N, Sommer JM, Newton A: An essential single domain response regulator required for normal cell division and differentiation in Caulobacter crescentus. EMBO J 1995, 14:3915-3924. 20. Wu J, Ohta N, Newton A: An essential, multicomponent signal transduction pathway required for cell cycle regulation in Caulobacter. Proc Natl Acad Sci U S A 1998, 95:1443-1448. 21. Matroule JY, Lam H, Burnette DT, Jacobs-Wagner C: Cytokinesis monitoring during development: rapid pole-to-pole shuttling of a signaling protein by localized kinase and phosphatase in Caulobacter. Cell 2004, 118:579-590. 22. Lam H, Matroule JY, Jacobs-Wagner C: The asymmetric spatial distribution of bacterial signal transduction proteins coordinates cell cycle events. Dev Cell 2003, 5:149-159. 23. Wheeler RT, Shapiro L: Differential localization of two histidine kinases controlling bacterial cell differentiation. Mol Cell 1999, 4:683-694. 24. Paul R, Jaeger T, Abel S, Wiederkehr I, Folcher M, Biondi EG, Laub MT, Jenal U: Allosteric regulation of histidine kinases by their cognate response regulator determines cell fate. Cell 2008, 133:452-461. This paper reports a positive feedback loop that ensures robust progression of the C. crescentus cell cycle. The authors find that the response regulator DivK promotes its own phosphorylation by stimulating the activity of its cognate histidine kinase DivJ and by switching its phosphatase PleC into the kinase mode, once a certain level of DivKP has accumulated in the cell. In addition, the PleC kinase activity is found to be required for the activation of the PleD, a response regulator with guanylate cyclase activity that is essential for the expression of adhesive properties and sessility. Current Opinion in Microbiology 2009, 12:715–721
25. Jacobs C, Hung D, Shapiro L: Dynamic localization of a cytoplasmic signal transduction response regulator controls morphogenesis during the Caulobacter cell cycle. Proc Natl Acad Sci U S A 2001, 98:4095-4100. 26. Sommer JM, Newton A: Turning off flagellum rotation requires the pleiotropic gene pleD: pleA, pleC, and pleD define two morphogenic pathways in Caulobacter crescentus. J Bacteriol 1989, 171:392-401. 27. Sommer JM, Newton A: Pseudoreversion analysis indicates a direct role of cell division genes in polar morphogenesis and differentiation in Caulobacter crescentus. Genetics 1991, 129:623-630. 28. Aldridge P, Paul R, Goymer P, Rainey P, Jenal U: Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus. Mol Microbiol 2003, 47:1695-1708. 29. Hecht GB, Newton A: Identification of a novel response regulator required for the swarmer-to-stalked-cell transition in Caulobacter crescentus. J Bacteriol 1995, 177:6223-6229. 30. Paul R, Abel S, Wassmann P, Beck A, Heerklotz H, Jenal U: Activation of the diguanylate cyclase PleD by phosphorylation-mediated dimerization. J Biol Chem 2007, 282:29170-29177. This study shows that the response regulator PleD dimerizes upon phosphorylation, leading to the stimulation of its guanylate cyclase activity and to its sequestration to the incipient stalked cell pole. 31. Paul R, Weiser S, Amiot NC, Chan C, Schirmer T, Giese B, Jenal U: Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain. Genes Dev 2004, 18:715-727. 32. Hengge R: Principles of c-di-GMP signalling in bacteria. Nat Rev Microbiol 2009, 7:263-273. 33. Purcell EB, Siegal-Gaskins D, Rawling DC, Fiebig A, Crosson S: A photosensory two-component system regulates bacterial cell attachment. Proc Natl Acad Sci U S A 2007, 104:18241-18246. This paper reports the identification of a two-component system that modulates the adhesive properties of C. crescentus in response to light. 34. Duerig A, Abel S, Folcher M, Nicollier M, Schwede T, Amiot N, Giese B, Jenal U: Second messenger-mediated spatiotemporal control of protein degradation regulates bacterial cell cycle progression. Genes Dev 2009, 23:93-104. This study identifies a novel c-di-GMP-binding protein, PopA that is required for degradation of the cell cycle regulator CtrA. PopA senses c-di-GMP with the help of a catalytically inactive guanylate cyclase domain. Upon ligand binding, it recruits CtrA to the differentiating pole, thus stimulating elimination of CtrA by the polar ClpXP protease complex. 35. Hinz AJ, Larson DE, Smith CS, Brun YV: The Caulobacter crescentus polar organelle development protein PodJ is differentially localized and is required for polar targeting of the PleC development regulator. Mol Microbiol 2003, 47:929-941. 36. Viollier PH, Sternheim N, Shapiro L: Identification of a localization factor for the polar positioning of bacterial structural and regulatory proteins. Proc Natl Acad Sci U S A 2002, 99:13831-13836. 37. Radhakrishnan SK, Thanbichler M, Viollier PH: The dynamic interplay between a cell fate determinant and a lysozyme homolog drives the asymmetric division cycle of Caulobacter crescentus. Genes Dev 2008, 22:212-225. This paper identifies the protein SpmX as a factor that required for the polar localization and activity of the histidine kinase DivJ, a key player in the regulatory network that governs the C. crescentus cell cycle. Synthesis and translation of the spmX mRNA occur at different developmental stages, controlled by a complex transcriptional cascade involving the cell cycle master regulator CtrA. SpmX carries a lysozyme-like domain that is necessary and sufficient for localization, suggesting that its localization is guided by structural cues in the cell wall. 38. Gorbatyuk B, Marczynski GT: Physiological consequences of blocked Caulobacter crescentus dnaA expression, an essential DNA replication gene. Mol Microbiol 2001, 40:485-497. 39. Collier J, Shapiro L: Feedback control of DnaA-mediated replication initiation by replisome-associated HdaA protein in Caulobacter. J Bacteriol 2009. www.sciencedirect.com
Spatial regulation in Caulobacter crescentus Thanbichler 721
This study demonstrates that, in C. crescentus, rapid inactivation of DnaA (RIDA) by the replisome-associated protein Hda is essential for suppressing supernumerary initiation events once the first round of replication has been started. 40. Jensen RB, Shapiro L: The Caulobacter crescentus smc gene is required for cell cycle progression and chromosome segregation. Proc Natl Acad Sci U S A 1999, 96:10661-10666. 41. Viollier PH, Thanbichler M, McGrath PT, West L, Meewan M, McAdams HH, Shapiro L: Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication. Proc Natl Acad Sci U S A 2004, 101:9257-9262. 42. Thanbichler M, Shapiro L: Chromosome organization and segregation in bacteria. J Struct Biol 2006, 156:292-303.
division, and polar organization in Caulobacter. Cell 2008, 134:956-968. Together with the work of Bowman et al. [50], this paper reports the identification of a coiled-coil protein, PopZ, that mediates attachment of the replication origin regions to the cell poles. PopZ assembles into a polymeric network that is sequestered to the tips of the cell and interacts with the origin-proximal ParB–parS nucleoprotein complex, a centromerlike structure involved in chromosome segregation. 52. Goehring NW, Beckwith J: Diverse paths to midcell: assembly of the bacterial cell division machinery. Curr Biol 2005, 15:R514-526. 53. Thanbichler M, Shapiro L: MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter. Cell 2006, 126:147-162.
43. Jensen RB, Wang SC, Shapiro L: A moving DNA replication factory in Caulobacter crescentus. EMBO J 2001, 20:4952-4963.
54. Wang SC, West L, Shapiro L: The bifunctional FtsK protein mediates chromosome partitioning and cell division in Caulobacter. J Bacteriol 2006, 188:1497-1508.
44. Shebelut CW, Jensen RB, Gitai Z: Growth conditions regulate the requirements for Caulobacter chromosome segregation. J Bacteriol 2009, 191:1097-1100.
55. Bigot S, Sivanathan V, Possoz C, Barre FX, Cornet F: FtsK, a literate chromosome segregation machine. Mol Microbiol 2007, 64:1434-1441.
45. Toro E, Hong SH, McAdams HH, Shapiro L: Caulobacter requires a dedicated mechanism to initiate chromosome segregation. Proc Natl Acad Sci U S A 2008, 105:15435-15440. This paper provides convincing evidence for the long-standing hypothesis that segregation of the origin regions is largely mediated by the ParAB DNA-partitioning system in C. crescentus. The binding sites for the centromer-binding protein ParB ( parS) are shown to impair the segregation process when introduced in trans. Moreover, using defined chromosome inversions and site-specific fluorescence labeling, the parS sites are identified as attachment points for the machineries mediating segregation and polar anchoring of the origin regions.
56. Huitema E, Pritchard S, Matteson D, Radhakrishnan SK, Viollier PH: Bacterial birth scar proteins mark future flagellum assembly site. Cell 2006, 124:1025-1037.
46. Mohl DA, Easter J Jr, Gober JW: The chromosome partitioning protein, ParB, is required for cytokinesis in Caulobacter crescentus. Mol Microbiol 2001, 42:741-755.
59. Aaron M, Charbon G, Lam H, Schwarz H, Vollmer W, Jacobs Wagner C: The tubulin homologue FtsZ contributes to cell elongation by guiding cell wall precursor synthesis in Caulobacter crescentus. Mol Microbiol 2007, 64:938-952. This study demonstrates that C. crescentus shows FtsZ-dependent medial growth, occurring independently of the MreB cytoskeleton. This observation is correlated with the recruitment of MurG, an enzyme involved in the synthesis of peptidoglycan precursors, to the cell division site.
47. Mohl DA, Gober JW: Cell cycle-dependent polar localization of chromosome partitioning proteins in Caulobacter crescentus. Cell 1997, 88:675-684. 48. Figge RM, Easter J, Gober JW: Productive interaction between the chromosome partitioning proteins, ParA and ParB, is required for the progression of the cell cycle in Caulobacter crescentus. Mol Microbiol 2003, 47:1225-1237. 49. Easter J Jr, Gober JW: ParB-stimulated nucleotide exchange regulates a switch in functionally distinct ParA activities. Mol Cell 2002, 10:427-434. 50. Bowman GR, Comolli LR, Zhu J, Eckart M, Koenig M, Downing KH, Moerner WE, Earnest T, Shapiro L: A polymeric protein anchors the chromosomal origin/ParB complex at a bacterial cell pole. Cell 2008, 134:945-955. (see comment on ref. [51]). 51. Ebersbach G, Briegel A, Jensen GJ, Jacobs-Wagner C: A self associating protein critical for chromosome attachment,
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57. Lam H, Schofield WB, Jacobs-Wagner C: A landmark protein essential for establishing and perpetuating the polarity of a bacterial cell. Cell 2006, 124:1011-1023. 58. Obuchowski PL, Jacobs-Wagner C: PflI, a protein involved in flagellar positioning in Caulobacter crescentus. J Bacteriol 2008, 190:1718-1729.
60. Divakaruni AV, Baida C, White CL, Gober JW: The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes. Mol Microbiol 2007, 66:174-188. Similar to the work of Aaron et al. [59], this paper shows the existence of a medial growth zone in C. crescentus. 61. Alyahya SA, Alexander R, Costa T, Henriques AO, Emonet T, Jacobs-Wagner C: RodZ, a component of the bacterial core morphogenic apparatus. Proc Natl Acad Sci U S A 2009, 106:1239-1244. This study reports the identification of a highly conserved membrane protein that mediates interaction between the MreB cytoskeleton and the cell wall biosynthetic machinery.
Current Opinion in Microbiology 2009, 12:715–721