CELL-CYCLE PROGRESSION AND THE GENERATION OF ASYMMETRY IN CAULOBACTER CRESCENTUS

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1 CELL-CYCLE PROGRESSION AND THE GENERATION OF ASYMMETRY IN CAULOBACTER CRESCENTUS Jeffrey M. Skerker and Michael T. Laub Microorganisms make tractable model systems and Caulobacter crescentus has emerged as the main model for understanding the regulation of the bacterial cell cycle. Mechanisms that mediate the generation and maintenance of spatial asymmetry are being uncovered using this model bacterium. Now, the advent of genomic technologies together with the completion of the Caulobacter crescentus genome sequence is enabling global analyses that have revolutionized the pace of research into the genetic networks that control the bacterial life cycle. G1 PHASE The period of time in the cell cycle before DNA replication starts and during which the cell contains only one copy of its genome. Bauer Center for Genomics Research, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA. Correspondence to M.T.L. laub@cgr.harvard.edu doi: /nrmicro864 Progression through the cell cycle, in all organisms, requires the precise coordination of four main processes: DNA replication, chromosome segregation, cell division and cell growth. The aquatic, non-pathogenic bacterium Caulobacter crescentus (which will be referred to as Caulobacter in this review) has emerged as the main model system for analysis of the prokaryotic cell cycle. This experimentally tractable organism can be investigated using a wide range of genetic, biochemical and cell biological techniques. The recent sequencing of the Caulobacter genome 1 has now enabled genomic approaches, such as DNA microarraybased expression profiling 2,3, and a variety of proteomic approaches 4 6.Significantly, Caulobacter cells are easily synchronized, thereby allowing precise temporal analysis of the cell cycle. Caulobacter has also emerged as a model system for understanding how bacteria establish and maintain cellular asymmetry. Each cell division in this organism produces two daughter cells that are morphologically and physiologically distinct (FIG. 1).Asymmetric cell division is a common feature in the life cycle of many bacteria 7.Perhaps the bestknown example is Bacillus subtilis, which, under certain starvation conditions, can divide asymmetrically as part of a developmental pathway that produces a spore. In addition, many bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Shigella flexneri and Listeria monocytogenes, might appear symmetric but have, on closer inspection, asymmetric poles. This article will review the progress that has been made in elucidating the molecular mechanisms underlying both cell-cycle progression and the establishment of asymmetry in Caulobacter. Recent studies have shown that many of the mechanisms discovered in Caulobacter are evolutionarily conserved among other members of the α-subdivision of proteobacteria. These species, including Sinorhizobium meliloti, Agrobacterium tumefaciens, Rickettsia prowazekii and Brucella abortus,have important roles in a wide range of environmental, medical and biowarfare protection applications 8 14 ; therefore, the fundamental research in Caulobacter has far-reaching implications. In particular, the identification of genes that are essential to cell-cycle progression in Caulobacter could help to identify new targets for antibacterial drug discovery. The cell cycle of Caulobacter crescentus: Progression through the Caulobacter life cycle (FIG. 1) requires the precise coordination of morphological, metabolic and cell-cycle events. The life cycle starts with a motile, chemotactic swarmer cell. This cell type has a single polar flagellum that is used for motility, and polar type IV pili that mediate adhesion to biotic and abiotic surfaces. The swarmer cell cannot initiate DNA replication and remains in G1 PHASE with a single NATURE REVIEWS MICROBIOLOGY VOLUME 2 APRIL

2 a Swarmer cell Stalked cell Early predivisional cell b DNA replication initiation S PHASE The period of time in the cell cycle in which a cell is actively synthesizing/replicating its genome. G2 PHASE The period of time in the cell cycle after DNA replication has been completed, but before cell division. DNA replication, nucleotide metabolism, recombination/repair Growth: cell envelope, ribosomes, oxidative respiration c Flagellum biogenesis Cell division Chemotaxis machinery DNA methylation Chromosome segregation Late predivisional cell G1 S G2 Pilus biogenesis Figure 1 Cell-cycle progression in Caulobacter crescentus.a Schematic of the cell cycle. Motile, piliated swarmer cells differentiate into stalked cells at the G1 S transition by shedding their polar flagellum, growing a stalk at that site, losing the polar pili and initiating DNA replication. Circles and θ structures in the cell represent quiescent and replicating chromosomes, respectively. An asymmetric predivisional cell yields two different progeny after division a swarmer and a stalked cell. The coloured bars indicate timing of gene transcription for functionally related sets of genes. Each set of genes shows just-in-time transcription: the genes are transcribed immediately before, or coincident with, the execution of the event for which they are required. Electron micrographs of a Caulobacter swarmer cell (b) and predivisional cell (c) prepared by negative staining with uranyl acetate. Scale bars equal 0.5 µm. chromosome. In response to cues that are not understood, the swarmer cell differentiates into a stalked cell (FIG. 1). During this differentiation, the polar flagellum is released and the polar pili are retracted. A stalk, which is a narrow elongation of the cell wall and membranes, is constructed at the same location as the discarded flagellum. Coincident with this morphological transition, the cell enters S PHASE and initiates DNA replication. Like most eukaryotes, but unlike many other prokaryotes such as E. coli, Caulobacter cells exhibit strict once-and-only-once replication behaviour; so, S phase produces exactly two daughter chromosomes. By contrast, rapidly growing E. coli cells can initiate DNA replication as many as four or more times between intervening cell divisions, such that daughter cells inherit a fully replicated chromosome that has already initiated several rounds of DNA replication. DNA replication and segregation of daughter chromosomes to opposite ends of the growing predivisional cell occurs during S phase and a brief, but distinct, G2 PHASE.Before dividing, the predivisional cell also builds a new flagellum and starts to construct new pili at the pole opposite the stalk. Once the flagellum is complete, cell division proceeds, yielding two daughter cells that are physiologically and morphologically different. One daughter cell is a stalked cell that immediately reinitiates another round of S phase and the other daughter cell is a swarmer cell that cannot start DNA replication until after the obligate swarmer-to-stalked cell differentiation step. Global approaches to cell-cycle study The elaborate spatial and temporal process of cellcycle progression in Caulobacter has been dissected at a molecular level using various genetic, biochemical and cell biological tools. More recently, the sequencing of the 4-Mb Caulobacter genome 1,encoding 3,767 genes, has provided a resource that has facilitated genomic approaches. Early work on Caulobacter led to the identification of more than 70 genes, many involved in DNA replication or flagellar biogenesis, that are transcriptionally regulated during the cell cycle. Whereas these genes were characterized one at a time, the completion of the genome sequence allowed the development and use of DNA microarrays for studying global patterns of gene expression 2,3.In the first global study 3,DNA microarrays were used to analyse changes in gene expression during cell-cycle progression. A population of Caulobacter cells were synchronized in the G1/swarmer phase and then allowed to proceed through the cell cycle, with RNA collected every 15 minutes and analysed on whole-genome DNA microarrays. The resulting expression profiles revealed a set of 553 genes more than 15% of the genome that are transcribed in a cell-cycle-dependent manner. The cell-cycle-dependent expression patterns of more than 70 genes that had been studied previously were confirmed by the DNA microarray data. Functional classification of these cell-cycle-regulated genes showed that Caulobacter couples gene expression to cell-cycle events with extraordinary precision; genes, the products of which are involved in a specific cell-cycle event, have a peak in expression immediately before, or coincident with, the timing of the event (FIG. 1).For example, transcription of the genes that are involved in DNA metabolism and DNA replication is induced and reaches a maximal level at the G1 S transition, whereas transcription of the genes that are involved in chromosome segregation is maximal in late S phase. This theme of just-in-time transcription applies to nearly all of the known cell-cycle 326 APRIL 2004 VOLUME 2

3 a Order of assembly Outer membrane Cell wall Periplasm Inner membrane PULSE CHASE STUDY A technique in which a cell, or cell extract, is briefly treated with a radioactive compound (the pulse ). This allows incorporation of the radiolabel into cellular constituents. The pulse is followed by addition of excess, non-radioactive compound (the chase ). Monitoring the radiolabelled compound over time then allows its location or stability to be tracked. Filament Basal body Hook Class IV Class III Class II Class I Figure 2 Transcriptional control of flagellar assembly. a The order of assembly of the Caulobacter flagellum proceeds from the inside to the outside of the cell, with class II components assembled first. Class II components include the inner-membrane secretion apparatus and the inner rings. This is followed by assembly of the class III components, which include two rings, the proximal and distal rod and the hook. The final step in the process is assembly of the filament, which is encoded by the class IV genes. b Clustered expression profiles of genes required for flagellar assembly 3, with cell-cycle progression running from left to right. Expression levels are colour-coded, with yellow indicating high mrna levels and blue representing low mrna levels. The order of gene transcription parallels the order of assembly. b events (FIG. 1).Precise transcriptional control might allow more efficient use of cellular resources and help to ensure the correct timing of events during the cell cycle. Close inspection of a subset of functionally related genes uncovered even more elaborate patterns of cellcycle transcription (FIG. 2).The genes that are required for flagellum assembly can be organized into four discrete classes (I IV) on the basis of their expression profiles. The order of expression of these four classes of genes was known from earlier work to parallel the order in which the gene products are assembled into a functioning flagellum, and the comprehensive DNA microarray analysis confirmed this temporal pattern. For a comprehensive review of flagellar assembly in Caulobacter, see REF. 15. Class I, which is expressed earliest, is composed of a single transcription factor, called CtrA (BOX 1), that sets the cascade of flagellar gene expression in motion and is the master regulator. This transcription factor activates expression of the class II genes, which encode components of the flagellum that are assembled in the inner membrane. Correct assembly of these components triggers the expression of the class III genes, which encode flagellar components that are assembled in the periplasm and outer membrane. Finally, after a short delay, the flagellar filament subunits (class IV genes), which comprise the flagellar filament, are expressed. The striking co-linearity of gene expression and organelle assembly occurs not Order of gene expression only with flagellum organelle assembly, but is also found for pilus biogenesis: transcription of the genes that encode components of the pilus temporally mirrors their order of assembly 3,16. An alternative global analysis of cell-cycle expression came from a large-scale study of protein synthesis rates using two-dimensional (2D)-gel electrophoresis and mass spectrometry 4.A strong correlation between the DNA microarray results and the 2D-gel data was found. However, there was a small group of genes for which the mrna levels did not correlate with protein synthesis, indicating that post-transcriptional regulation of these genes occurs. Finally, a large-scale PULSE CHASE STUDY 4 identified 48 proteins that have extremely short half-lives. Twenty-six of these proteins also showed cell-cycle-regulated expression, indicating that the abundance, and presumably the activity, of these proteins is precisely controlled during the cell cycle. This set of 26 proteins includes several essential proteins that had previously been shown to be degraded at specific times during the cell cycle Caulobacter, like eukaryotic cells, seems to use regulated proteolysis as a mechanism for regulating the cell cycle and ensuring irreversible transitions during its development. CtrA regulation. How are the intricate gene-expression patterns that are observed during the Caulobacter cell cycle coordinated and regulated? Previous work has identified a key master regulator, CtrA, which is essential for viability and controls a large number of cell-cycle events 20 (BOX 1).CtrA is a member of the highly conserved, ubiquitous two-component signal-transduction family, comprised of histidine kinases and their substrates, response regulators (BOX 2).CtrA, a response regulator, contains a DNA-binding domain, and phosphorylation of CtrA at a single site increases its affinity for target promoters 21.Phosphorylated CtrA (CtrA~P) functions as a transcriptional activator and repressor during the cell cycle. Like master regulators in many organisms, CtrA is itself subject to multiple levels of regulation (FIG. 3), which help to ensure the fidelity of cell-cycle progression 17,20,22 27.CtrA~P is present at high concentrations in swarmer/g1 cells. Rapid proteolysis a ClpXPdependent process eliminates CtrA at the swarmerto-stalked cell transition 17,22.The proteolysis of CtrA, which binds with high affinity to five sites in the origin of replication to repress chromosome replication, allows DNA replication to begin 28.By mechanisms that are not fully understood, the commencement of S phase leads to an initiation in transcription of ctra from the promoter P1. As the concentration of CtrA increases, CtrA binds to, and represses, transcription from P1, but strongly activates transcription from promoter P2 (REF. 29).This positive feedback results in a rapid accumulation of newly synthesized CtrA, with estimates of production of ~22,000 molecules per cell 30.Newly synthesized CtrA is phosphorylated, which enables it to regulate the expression of its 95 target genes. In the stalked half of the late predivisional cell, CtrA is again degraded in a ClpXP-dependent NATURE REVIEWS MICROBIOLOGY VOLUME 2 APRIL

4 Box 1 The master regulator CtrA CtrA (cell-cycle transcription regulator) A genetic screen for essential genes that activate the flagellar hierarchy identified CtrA as a transcription factor controlling multiple cell-cycle processes. The master regulator CtrA coordinates the timing of cellular events with chromosome replication. Important features of CtrA include: it is essential for viability it is a member of the two-component signal-transduction family of proteins functions as a response regulator (BOX 2) it is conserved in many species of the α-subdivision of proteobacteria it binds to the chromosomal origin of replication to repress DNA replication initiation it functions as a transcription factor to directly regulate 95 genes involved in polar morphogenesis, cell-cycle progression and regulatory processes its activity during the cell cycle is controlled on at least three partially redundant levels: transcription, proteolysis and phosphorylation. manner. Cell division results in a swarmer cell that contains high concentrations of phosphorylated CtrA, and a stalked cell that contains little or no CtrA. The asymmetric distribution of phosphorylated CtrA is a crucial step in establishing daughter cells that have distinct fates in Caulobacter. The regulation of CtrA is controlled at the level of transcription, proteolysis and phosphorylation. Below, we review the recent progress that has been made in revealing the details of each of these control mechanisms, as well as initial evidence that subcellular localization might also have a role in regulating CtrA function. Transcription of ctra. DNA methylation has recently been shown to have an important role in the activation of the ctra P1 promoter in stalked cells. The P1 promoter, which contains a DNA methylation site between the 10 and 35 regions, is sensitive to methylation, such that it is more active in a hemi-methylated or unmethylated state 23.At the onset of S phase, the chromosome, including the ctra locus, is fully methylated and the P1 promoter is inactive. During chromosome replication, the replication fork passes the ctra locus and, because newly synthesized DNA is not immediately methylated, the ctra loci in the new chromosomes are hemi-methylated. At this point, P1 becomes transcriptionally active. Consistent with this model, moving the ctra locus closer to the chromosome replication terminus delays transcription from P1. Once CtrA accumulates at high concentrations in the predivisional cell, it activates transcription of ccrm,which encodes a DNA methyltransferase that fully methylates the daughter chromosomes. This renders the P1 promoter of both new copies of the ctra locus less transcriptionally active. The precise mechanism by which the change in methylation status of P1 affects transcription is unclear. One likely possibility is that the transition from a fully methylated to a hemi-methylated state increases the affinity of the promoter for an as-yet-uncharacterized transcription factor. DNA METHYLATION The addition of a methyl (CH 3 ) group to adenine or cytosine bases in DNA. Box 2 Two-component signal transduction. Two-component signal transduction systems are widespread in bacteria, fungi and plants, and are used by these organisms to respond to various environmental and intracellular stimuli 75,76.The two components are sensor histidine kinases and response regulators. As shown in the figure (part a), activation of a histidine kinase leads to autophosphorylation on a conserved histidine residue. The phosphoryl group is then typically transferred to an aspartate residue that is located on the receiver domain of a response regulator, thereby activating it; response regulators are frequently transcription factors, and phosphorylation renders them competent to bind DNA and regulate the transcriptional output of target genes. Multicomponent phosphorelays also exist (see figure part b), in which the phosphoryl group is shuttled from a histidine kinase, to a response regulator, to an intermediate protein known as a histidine phosphotransferase and then to a final response regulator. An important subclass of the histidine kinases are the hybrid kinases, which are histidine kinases with a carboxy-terminal response-regulator domain. a Input signal Response regulator H D~ P Histidine kinase H~ P P ~H b Histidine kinase P ~H P ~D Input signal H Response regulator Histidine phosphotransferase 328 APRIL 2004 VOLUME 2

5 CtrA~P CtrA proteolysis ctra P1 transcription ctra P2 transcription CtrA phosphorylation P1 HOMOLOGY MODELLING A procedure in which an unknown protein structure is modelled by matching fitting to the known structure of a closely related protein by matching conserved amino acids. Allows an approximation of the 3D shape and organization of the protein to be obtained. P2 ctra Gene CtrA ~ P expression Figure 3 Regulation of the master regulator CtrA. Coloured bars below a diagram of the Caulobacter cell cycle indicate the timing of CtrA proteolysis, transcription and phosphorylation. Active, phosphorylated CtrA protein (pink shading) is present in the swarmer cell and the early predivisional cell. CtrA is specifically degraded at the swarmer-to-stalked cell transition, and in the late predivisional cell after compartmentalization 22. ctra transcription is initiated at two promoters 29, P1 and P2, shown at the bottom of the figure. The weak P1 promoter is activated first in the early predivisional cell. The initial accumulation of active CtrA leads to negative feedback on promoter P1, and leads to strong positive feedback at the P2 promoter. The net result is the rapid accumulation in the predivisional cell of CtrA~P, which is required for a number of essential cellcycle events. Even if CtrA is present throughout the cell cycle, regulated phosphorylation ensures cell-cycle control of CtrA activity 22. CtrA Proteolysis and compartmentalization of CtrA. Precise temporal and spatial control of CtrA proteolysis helps to regulate cell-cycle progression and the production of asymmetric daughter cells. Cell-cycle-regulated proteolysis of CtrA is dependent on the ClpXP protease 17.Depletion of either of the two Clp subunits (ClpX or ClpP) in vivo leads to stabilization of CtrA, which in turn results in a defect in the G1 S transition, presumably because CtrA remains bound to the origin of replication. A second essential response regulator, DivK also contributes to CtrA proteolysis. Cells that have a cold-sensitive mutant allele of divk do not degrade CtrA at the G1 S transition at the non-permissive temperature 25.CtrA is not degraded in vitro by purified, active ClpXP and phosphorylated DivK 25,which might indicate either that DivK control of CtrA proteolysis is indirect, or that another necessary proteolysis factor remains to be found. In E. coli, ClpXP-mediated proteolysis often requires substrate-specificity factors that target particular substrates for degradation 31,32,which supports the hypothesis that another factor is required. The ClpXP protease typically recognizes hydrophobic residues at the carboxyl terminus of substrate proteins. Deletion of the C-terminal three residues of CtrA, or modification of the last two residues from Ala Ala to Asp Asp, results in a proteolytically stable form of CtrA 22. Although the C-terminal portion of CtrA is necessary for proteolysis, it does not seem to be sufficient to trigger degradation. However, the amino-terminal 56 residues of CtrA also known as the receiver domain (RD) together with the 15 C-terminal residues (RD+15) were shown to be sufficient for cell-cycle-regulated proteolysis of CtrA 24.To delineate those residues that are necessary for ClpXP-mediated proteolysis of CtrA, Ryan et al. 24 fused the RDs from five separate CtrA homologues (from closely related species in the α-subdivision of proteobacteria) to the last 15 C-terminal residues of the Caulobacter CtrA. Four out of the five hybrid proteins, when expressed in Caulobacter,were degraded temporally with a pattern that nearly matches that of native CtrA, indicating that the RDs of these four proteins contain a common proteolytic signal. By contrast, the hybrid protein that contained the RD of CzcR, the CtrA homologue from Rickettsia prowazekii, was stable throughout the cell cycle. A sequence alignment of the five CtrA RDs identified 10 amino acids that are conserved in all except CzcR, which makes these sites good candidates for forming part of the proteolytic signal. HOMOLOGY MODELLING of the Caulobacter CtrA RD using a solved structure of the RD of B. subtilis Spo0F predicts that 9 of these 10 residues are on one surface of the protein and could form a binding surface for a factor that regulates the temporal pattern of CtrA proteolysis. This factor is unlikely to be a subunit of the ClpXP protease, because clpx and clpp are constitutively expressed and are present throughout the cell cycle 17. The Caulobacter CtrA RD+15 is sufficient to target heterologous proteins that are unrelated to CtrA for degradation, presumably by ClpXP. For example, a fusion of yellow fluorescent protein (YFP) to the CtrA RD+15 (YFP RD+15), was sufficient to confer a cell-cycle-regulated degradation pattern on YFP 24. This YFP construct allows direct visualization, by fluorescence microscopy, of protein turnover in vivo. As expected, the YFP RD+15 construct was degraded during the swarmer-to-stalked cell transition and was degraded in the portion of the predivisional cell that was destined to become the stalked cell. Surprisingly, a significant percentage of cells localized the fluorescent fusion to the flagellar pole of the swarmer cell immediately prior to degradation at the swarmer-to-stalked transition. The functional significance of this remains to be shown, but it seems likely that subcellular localization is coupled to degradation. How is the proteolysis of CtrA confined to only one portion of the predivisional cell? Using the YFP RD+15 construct and fluorescence microscopy, Judd et al. 30 showed that CtrA proteolysis occurs in the stalked half of the predivisional cell only after septum formation, which presumably prevents free diffusion of CtrA between the two nascent daughter cells. This indicates that septum formation is coupled to the initiation of proteolysis, but the mechanism of this coupling remains uncharacterized. Phosphorylation of CtrA. Phosphorylation is perhaps the most important, but least well-understood, aspect of CtrA regulation. CtrA only becomes competent to activate transcription after phosphorylation of the amino acid residue Asp51 (REF. 20). Several two-component NATURE REVIEWS MICROBIOLOGY VOLUME 2 APRIL

6 Box 3 A beginner s guide to Caulobacter gene nomenclature Ple (pleiotropic phenotype) A screen for mutants with defects in multiple processes, including motility, phage sensitivity and stalk formation, yielded the two-component signalling genes plec and pled. Div (division phenotype) A screen for suppressors of the plec pleiotropic phenotype yielded three two-component signalling genes, divj, divk and divl; mutations in each individually yield a cell-division phenotype. Cck (cell-cycle kinase) A screen for temperature-sensitive, lethal, pleiotropic mutants identified the kinase CckA. A yeast two-hybrid screen for proteins that interact with DivK identified two other histidine kinases that might be involved in the cell cycle, CckN and CckO. systems have been identified that have roles in controlling the phosphorylation state of CtrA (FIG. 4). One of the proteins that regulates CtrA, the histidine kinase CckA, is essential for viability, and the phenotype of a ccka mutant is similar to that of a ctra mutant 33 (BOX 3). Also, DNA-microarray analysis has shown that ctra and ccka mutants have almost identical expression profiles 34.Perhaps the strongest evidence for the role of CckA in the regulation of CtrA is that a temperaturesensitive ccka mutant contains little to no phosphorylated CtrA 33.CckA is also phosphorylated in a temporal cell-cycle pattern that closely matches that of CtrA 34. These data indicate that an important role of CckA is to phosphorylate CtrA, but it is not known whether the effect is direct or indirect. CckA is a hybrid histidine kinase, which is a fusion between a kinase and a response regulator (BOX 2).Hybrid histidine kinases often participate in MULTI-COMPONENT PHOSPHORELAYS,in which a histidine phosphotransferase (Hpt) protein Histidine kinase DivJ PleC CckN CckO DivL CckA Response regulator Histidine phosphotransferase Response regulator DivK Hpt CtrA Figure 4 Phosphorylation of the essential response regulators CtrA and DivK. The figure shows a model for the signal transduction pathways that affect DivK and CtrA phosphorylation. Solid arrows indicate pathways that are supported by direct experiments and dashed arrows show proposed or hypothetical pathways. There is evidence for at least five histidine kinases that bind to and/or influence the phosphorylation state of the essential response regulator DivK. DivJ has been shown to phosphorylate DivK in vitro 37. PleC seems to function as a phosphatase for DivK~P 37,65. CckN, CckO and DivL have been found to interact with DivK in a two-hybrid screen 36. Although DivJ and DivL can both phosphorylate CtrA in vitro 27, only CckA has been shown to be required for CtrA phosphorylation in vivo 33. An allele of ctra was identified that can suppress mutations in the three div genes and plec 26, suggesting that all five components lie in the same pathway. This might involve an unidentified histidine phosphotransferase (Hpt), which shuttles a phosphoryl group from DivK to CtrA, both response regulators. CckA is a hybrid kinase (BOX 2), which also raises the possibility of a Hpt-mediated pathway that controls CtrA phosphorylation. Hpt transfers a phosphoryl group from the hybrid kinase to a second response regulator. This raises the possibility that a Hpt protein functions in Caulobacter to mediate signal transduction between CckA and CtrA. The kinases PleC, DivJ and DivL, and the response regulator DivK, are also thought to help mediate the phosphorylation state of CtrA 26,27.DivJ, DivK and DivL were each identified as genetic suppressors of plec 35,and an allele of ctra was subsequently found that was a suppressor of the three mutant div alleles 26,which suggests that all of these genes function in the same pathway. More recently, a yeast two-hybrid analysis, which used DivK as the bait, showed that DivK interacts not only with DivJ, PleC and DivL, but also with two other uncharacterized kinases, CckN and CckO 36.Using purified proteins, direct biochemical links have been established in vitro for some of these two-component proteins 26,27,37,but the in vivo significance of these interactions remains unclear (FIG. 4).A complete understanding of the complex pathways controlling CtrA phosphorylation will ultimately require more detailed genetic and biochemical analysis, similar to that needed for elucidating other complex two-component signalling pathways, such as the phosphorelay pathway that leads to activation of Spo0A, the master regulator of sporulation in B. subtilis 38. Mapping the CtrA regulon The complex regulation of CtrA activity, along with the essential nature of the ctra gene, indicates that this transcription factor has an important role in controlling cell-cycle patterns of gene expression. Early work on CtrA showed that it transcriptionally controls about a dozen genes, the products of which participate in cell-cycle events 16,18,20,39,40. More recently, whole-genome DNA microarray analyses have provided a complete description of the CtrA regulon 2,3 (FIG. 5).Global expression profiles of a Caulobacter strain with a temperature-sensitive allele of ctra were obtained using microarrays. In total, 144, or 26% of all 553 cell-cycle-regulated genes, were significantly affected by shifting to the restrictive temperature and the subsequent loss of CtrA function 3 ; this included 84 genes that are activated by CtrA and 60 genes that are repressed by CtrA. Expression profiling alone is insufficient to determine whether these regulatory effects are direct, but a technique known as location analysis (BOX 4) allows mapping of the DNA binding sites of a protein in vivo on a genome-wide scale 41,42.This technique was used to identify the 95 genes in 55 operons that have regulatory regions bound by CtrA, and that also showed significant changes in transcript levels in the ctra mutant strain. These 95 directly regulated genes comprise the CtrA regulon 2.Twenty-nine of these genes are repressed by CtrA most of these genes show maximal expression during the G1 S transition, which is precisely when CtrA has been degraded and therefore depleted from the cell. Sixty-six genes are activated by CtrA. Although most of these genes are expressed after accumulation of CtrA during S phase, their peak induction times span a 60-minute 330 APRIL 2004 VOLUME 2

7 P1 P2 ctra CtrA CtrA binding site and promoter CtrA ~ P Regulatory pathways PilA Pilus biogenesis Cell-cycle regulation pila Che Chemotaxis machinery 8 regulatory genes Cell-cycle regulation che (14 genes) DivK 3 regulatory genes Fla Flagellum biosynthesis divk ClpP CtrA proteolysis fla (16 genes) CcrM DNA methylation clpp HfaAB Holdfast synthesis ccrm FtsZ hfaab? ftsz Fts Cell division, cell-wall synthesis 39 unknown genes Chromosome replication fts (ftsq, ftsw, murg) Origin of replication Figure 5 CtrA regulon. The master regulator CtrA controls many cell-cycle events. Phosphorylated CtrA (CtrA~P) directly activates or represses the transcription of 95 genes 2, which constitute the CtrA regulon. These include genes that are required for essential cell-cycle processes, such as cell division (ftsz) and DNA methylation (ccrm). CtrA~P also controls polar morphogenesis by activating the flagellar biogenesis cascade, activating the transcription of the pila gene, which encodes the pilin subunit, and activating the transcription of genes that are needed for holdfast synthesis (hfaab). CtrA~P blocks chromosome replication in the swarmer cell by binding to five sites in the origin of replication 28. CtrA~P also regulates a number of other regulatory genes, including two (divk and clpp) that are involved in cell-cycle-regulated proteolysis of CtrA. MULTI-COMPONENT PHOSPHORELAY A signalling pathway involving two-component signal transduction molecules, in which a phosphoryl group from ATP is transferred to more than two components. Usually involves transfer from a histidine kinase to a response regulator to a histidine phosphotransferase to another response regulator. CLOSED-LOOP SET A set of regulatory factors that regulate each other such that the overall topology produces a circle, or loop, of interactions. period. This raises the question of how the timing of expression of CtrA-activated genes can vary so much. One possibility is that the affinities of different promoters for CtrA vary significantly, affecting the timing of gene expression 39.Alternatively, additional transcription factors, or alternative sigma factors, could fine-tune the timing of different CtrA-dependent genes. The functions of CtrA-regulated genes can be divided into three main classes 2 : first, genes that are required for polar morphogenesis, including flagellum and pilus biogenesis; second, genes that are essential for cell-cycle processes, such as cell division and DNA methylation; and third, regulatory genes (FIG. 5). CtrA directly controls expression of 11 regulatory genes, which indicates that the Caulobacter cell cycle might be controlled, at least in part, by a cascaded, CLOSED-LOOP SET of regulatory genes, similar to that proposed for the yeast cell cycle 43.It is of particular interest to note that two members of the CtrA regulon 2, divk and clpp,are involved in the cell-cycle-regulated proteolysis of CtrA. Positive transcriptional activation of divk and clpp by CtrA, followed by negative feedback in the form of DivK/ClpXP-mediated CtrA proteolysis, might form the basis of an oscillator that underlies cell-cycle progression in Caulobacter. Cell cycle: dependencies and checkpoints Cell-cycle progression in Caulobacter requires the precise temporal coordination of cellular and morphological events, and seems to be accomplished largely by the organization of cell-cycle events into a series of dependent relationships. In particular, cell division is known to be dependent on successful DNA replication 44 and the completion of chromosome segregation. But what is the molecular basis for these critical dependencies? Hartwell and Weinert 45 proposed that similar dependent events in the eukaryotic cell cycle could be coupled by either of two distinct mechanisms: substrate product relationships and checkpoints. We will briefly review each of these mechanisms and then discuss what is known about their role in coupling dependent processes during the cell cycle and during morphogenesis in Caulobacter. NATURE REVIEWS MICROBIOLOGY VOLUME 2 APRIL

8 Box 4 DNA binding site analysis by immunoprecipitation and DNA microarrays The figure shows a schematic diagram of location analysis, a procedure that is used to identify in vivo binding sites for transcription factors on a genomewide scale 2,41,42.Cells are treated with formaldehyde to crosslink CtrA to its in vivo binding sites 2. Genomic DNA is extracted, fragmented and then subjected to immunoprecipitation (IP) with an anti-ctra antibody. A mock IP is performed without antibody as a control. The crosslinks are reversed, and the immunoprecipitated DNA is amplified by ligation-mediated PCR. The DNA amplified from the IP is labelled with the fluorophore Cy5 (shown in red) and, using a DNA microarray, is compared with DNA amplified from the mock IP that is labelled with the CtrA CtrA-bound promotor Crosslink CtrA to DNA, extract and shear DNA IP with CtrA-specific antibody Reverse crosslinks, amplify and label by PCR IP without antibody (mock IP) fluorophore Cy3 (shown in green). DNA targets bound in vivo by CtrA should be enriched in the CtrA IP sample and the corresponding spots will appear more red (high Cy5:Cy3 ratio) on the array. Location analysis can be done with any protein for which an antibody is available, and has been used to study binding of transcription factors in a number of organisms 41,43,77.Figure reproduced with permission from REF. 2 (2002) National Academy of Sciences, USA. The substrate product mechanism is exemplified by bacteriophage T4 baseplate morphogenesis 46.The T4 baseplate is composed of six identical wedges, each containing seven different proteins. The assembly of the wedge occurs in a precisely ordered pathway (FIG. 6a). Each step in the assembly process forms an essential substrate for the addition of the next subunit. The order of assembly is controlled by the physical chemistry of protein protein interactions, and by the conformational changes that occur with proper assembly 46.This example shows that a complex process can be precisely ordered without the need for any extrinsic factors; the components of the process itself have an intrinsic set of dependent interactions. Alternatively, a checkpoint mechanism could exist to ensure the proper execution of cell-cycle events. Hartwell and Weinert 45 originally defined a cell-cycle checkpoint as a dedicated surveillance mechanism that, in response to a failure to complete event A, negatively regulates event B. Such a system is extrinsic to the events themselves, because the components of the checkpoint system are not involved in the execution of either event. The key to distinguishing a checkpoint mechanism from a substrate product relationship is the identification of a mutation that gives relief of inhibition 45.A mutation in an extrinsic, checkpoint system would relieve the inhibition that is imposed on event B by the failure of event A. For substrate product relationships, no such mutation can be found, because the dependency is caused entirely by factors intrinsic to events A and B. The SOS response is one example of a cell-cycle checkpoint in a bacterial system 47 (FIG. 6b).After exposure to ultraviolet (UV) irradiation, or other DNA damaging agents, E. coli cells are blocked for cell division until the damage is repaired, which ensures that any progeny cells do not inherit a damaged chromosome; cell division is therefore considered to be dependent on successful DNA replication. In response to DNA damage, E. coli rapidly synthesizes the protein SulA, which functions to specifically block cell division by binding to FtsZ, an essential component of the cell division machinery. By contrast, a sula mutant does not properly arrest the cell cycle when DNA damage occurs, because without SulA, FtsZ, and therefore cell division, cannot be inhibited. The sula mutation provides relief of inhibition 48.SulA is not normally required for DNA replication or cell division. It acts extrinsically to both processes, and ensures that their dependent relationship is maintained in perturbed conditions; it constitutes a true checkpoint system. Are there cell-cycle checkpoints in Caulobacter? Although Caulobacter does not have a sula homologue, it does couple DNA replication and cell division 49,50.Treatment of Caulobacter cells with DNA damaging agents transiently blocks cell division. In response to DNA damage, transcription from the P2 promoter (FIG. 3) of ctra is rapidly reduced 50.This leads to a decrease in the concentration of active, phosphorylated CtrA in the predivisional cell, which in turn prevents transcription of two CtrA-dependent genes, ftsq and ftsa,which are required for the final stages of cell division. This establishes a mechanistic basis for coupling DNA damage to the inhibition of cell division. However, no mutations 332 APRIL 2004 VOLUME 2

9 a T4 baseplate wedge assembly gp11 gp6 gp DNA damage sensed gp7 gp gp b Escherichia coli DNA damage response DNA damage checkpoint DNA replication SOS response Cell division cycle SulA Cell division Figure 6 Strategies for coupling-dependent processes. Dependent processes can be controlled by either a substrate product or checkpoint mechanism. a Morphogenesis of the T4 baseplate wedge is a classic example of a substrate product relationship 46. Assembly of the wedge occurs in an invariant order by the successive addition of subunits. For example, gp7 can bind only to the gp10 dimer, and gp8 cannot bind to gp7 unless the latter is preassembled into a gp10 gp7 complex. These dependencies ensure that wedge assembly follows an invariant pathway (gp10, gp7, gp8, gp6, gp53 and gp25). If any of the subunits are missing, baseplate assembly is blocked. Addition of gp11 can occur at any time, so it is not part of the dependency pathway. b The SOS response in Escherichia coli is an example of a bacterial cell-cycle checkpoint 47, as described in the text. During an unperturbed cell cycle (blue box), cell division is dependent on DNA replication and undamaged DNA. In response to DNA damage, the checkpoint system is activated (orange box). The checkpoint surveillance mechanism is extrinsic to the process it controls and is dispensable in a normal cell cycle. Detection of DNA damage activates the SOS response, resulting in production of the SulA protein. SulA inhibits the polymerization of FtsZ, which is an essential cell division protein, and cells arrest until the DNA damage is repaired. Mutations in sula relieve this inhibition. 11 have yet been found that give relief of inhibition, which is the gold standard for identifying a checkpoint. Hypothetically, the identification of relief-of-inhibition mutations would reveal genes that function in a dedicated surveillance system that monitors DNA damage, and transmits an inhibitory signal to regulators of, or essential components of, the cell-cycle machinery, such as CtrA. Another dependency relationship in the Caulobacter cell cycle that could be governed by a checkpoint mechanism is the requirement for completion of chromosome segregation before cell division. Several highly conserved genes, including para, parb, parc, pare and smc in Caulobacter,have been shown to have essential roles in chromosome segregation Depletion of any one of these proteins prevents chromosome segregation, which leads to a block of cell division. This dependency ensures that each daughter cell inherits a full complement of the Caulobacter genome, but no mutation has yet been described that gives relief of inhibition, leading to an attempt at cell division without the completion of chromosome segregation. Such a mutation would help to identify a dedicated, extrinsic surveillance system that monitors the status of chromosome segregation, if one exists. Alternatively, the coupling of chromosome segregation to cell division might be driven by a mechanism that is more similar to the substrate product mechanism described above. In fact, in E. coli, the interruption of chromosome segregation and the physical presence of DNA at the mid-cell site, prevents the cytokinesis machinery from executing cell division, a mechanism called nucleoid occlusion 55.Whether Caulobacter operates in a similar manner or uses a dedicated checkpoint system remains to be shown. Coupling morphological events to gene expression. There are several well-studied examples in Caulobacter that show that gene expression is dependent on the completion of a morphological event, most of which function to ensure the proper assembly of the polar flagellum. This complex organelle is composed of more than 30 gene products, and as described before, these gene products are arranged in a four-tiered cascade (class I IV), in which the order of gene expression parallels the order of flagellum assembly (FIG. 2). The expression of the genes in each class depends on the expression and assembly of the preceding class. These so-called morphological couplings 56 seem to involve dedicated surveillance machinery, but are intrinsic to the system, and therefore incorporate aspects of both of the coupling mechanisms described by Hartwell and Weinert. A defect in the expression or assembly of a class II flagellar component prevents the transcription of class III and class IV genes. Mutants were identified that restore class III gene expression in a class II mutant 57. These bypass of flagellar assembly (bfa) mutants are dominant mutations in two regulatory genes, flbd, and flix FliX is not a structural component of the flagellum, but instead seems to transmit the state of flagellar assembly to FlbD, which is a transcription factor. FliX, by an unknown mechanism, keeps FlbD inactive until NATURE REVIEWS MICROBIOLOGY VOLUME 2 APRIL

10 NASCENT SWARMER POLE The pole opposite the stalked pole in a Caulobacter predivisional cell, where the flagellum and pilus secretion apparatus must be assembled before cell division takes place. class II gene products have properly assembled. FlbD is then activated, class III gene expression, and therefore flagellar assembly, continues. Neither flix nor flbd are part of the flagellum they seem to be dedicated to surveillance of flagellar assembly and so are reminiscent of a checkpoint system. But both FliX and FlbD are required every time a flagellum is built in Caulobacter, so they are intrinsic to the assembly process, in contrast to canonical cell-cycle checkpoint systems that are required when a process malfunctions, but are otherwise dispensable. Morphological coupling also occurs between class III and class IV flagellar genes: mutations in class III genes prevent the production of flagellar filament monomers that are encoded by the class IV genes. The FlbT protein binds to a 5 untranslated region of class IV mrnas and facilitates their degradation By a mechanism that is not yet understood, the proper assembly of flagellar subunits encoded by class III genes prevents FlbT-mediated degradation of class IV flagellin mrnas. This, in turn, leads to synthesis of the flagellin monomers and, ultimately, to their assembly into a functioning flagellar filament. In common with flix and flbd, flbt seems to be dedicated to the surveillance of proper flagellar assembly, but it is intrinsic to the process because it is required each time the organism assembles a flagellum. The FlbT-based coupling of class III and class IV flagellar genes therefore has elements of both a classical checkpoint system and a substrate product mechanism. Subcellular protein localization In addition to the elaborate circuitry that controls temporal progression through the cell cycle, every round of cell division requires the spatial organization of an asymmetric predivisional cell to produce two different daughter cells. From work with in vivo fluorescent reporters, such as green fluorescent protein (GFP), it has become clear in recent years that in many bacteria, subcellular localization of regulatory proteins can contribute to these spatial patterning processes bacteria can no longer be perceived as simple bags of enzymes 7,64. The subcellular localization of three Caulobacter histidine kinases (CckA, DivJ and PleC) is important for the production of daughter cells that have distinct fates (FIG. 7). In the predivisional cell, CckA and PleC localize predominantly at the NASCENT SWARMER POLE,whereas DivJ localizes almost entirely at the stalked pole 33,65.CckA is necessary for the phosphorylation and activation of the master regulator CtrA. The localization of CckA to the swarmer cell compartment is consistent with the observation that swarmer cells contain increased concentrations of CtrA~P compared with stalked cells. Therefore, it seems that the localization and asymmetric distribution of the CckA kinase helps to establish an important difference in daughter cell fate: new swarmer cells cannot initiate DNA replication because high concentrations of CtrA~P repress the origin of replication, whereas new stalked cells, with little to no CtrA~P, are free to initiate S phase. DivJ and PleC also help to establish different cell fates in the two daughter cells. DivJ is a kinase that phosphorylates two response regulators, DivK and PleD. By contrast, PleC has demonstrated phosphatase activity for DivK~P, and has recently been shown in vivo to directly or indirectly inhibit the formation of PleD~P, possibly as a phosphatase 26,27,37,66.Taken together, these data indicate that the localization of DivJ and PleC to opposite poles 65 stalked and swarmer, respectively of the late predivisional cell helps to ensure high levels of phosphorylated DivK (DivK~P) and phosphorylated PleD (PleD~P) only in the new stalked cell. The asymmetric distribution of DivJ and PleC might therefore help to determine daughter cell fates: active DivK is required for CtrA degradation 25, so its presence in the stalked portion of septated predivisional cells presumably targets CtrA for degradation and thereby triggers initiation of DNA replication in this daughter cell. Conversely, without active DivK in the nascent swarmer cell, CtrA will not be degraded and will repress the initiation of DNA replication. By a mechanism that is not yet understood, phosphorylated PleD triggers flagellar release at the swarmer-to-stalked-cell transition 67,68,so the inheritance of PleC to the swarmer cell probably helps to prevent flagellar release in that cell type as PleC inhibits the formation of PleD~P. Differentiation into a stalked cell, and the localization of DivJ to the stalked pole, could therefore initiate PleD-mediated flagellar release. Localization of PleC to the swarmer pole is also critical for the proper assembly of pili only in swarmer cells 69,70. PleC is required for accumulation of the pilin subunit PilA, which polymerizes into the pilus filament. Interestingly, cells containing an active, but delocalized, PleC fail to accumulate PilA, which provides the first evidence in Caulobacter that precise subcellular localization of a kinase in the membrane is required for kinase activity 71. PodJ is at the top of a localization hierarchy. How are these kinases targeted to, and retained at, a specific cell pole? Little is known about the localization of CckA or DivJ, but a localization determinant for the kinase PleC has recently been identified 71.This factor, which is encoded by the podj gene, is a large, membraneassociated protein that is localized to the nascent swarmer pole in predivisional cells. PodJ is required for the correct localization of PleC to the swarmer pole, and also specifies the localization of CpaC and CpaE two membrane-bound proteins that are involved in pilus biogenesis. After localizing to the nascent swarmer pole, and directing the localization of PleC, CpaC and CpaE, PodJ is processed by an unknown protease to a shorter form that is retained at the swarmer pole, although the functional consequences of this processing are not understood 71,72.Full-length PodJ is predicted to contain a long, α-helical region at the N-terminus 73, and has motifs that are a signature of coiled-coils a structural motif that is often involved in protein protein interactions. PodJ could interact with itself through this region, potentially providing a structural scaffold for the assembly of other proteins, such as PleC. PodJ might also localize other proteins through its predicted 334 APRIL 2004 VOLUME 2

11 PleC CckA PleC CckA PleC CckA CtrA ~ P CtrA ~ P CtrA ~ P DivK ~ P PleD DivK PleD DivK CtrA ~ P PleD DivK PleC CckA DivK ~ P PleD ~ P DivJ PleD ~ P DivK ~ P PleD ~ P CckA DivJ DivK ~ P PleD ~ P CckA DivJ DivJ Figure 7 Subcellular localization and cellular asymmetry. Asymmetry in the Caulobacter predivisional cell is determined largely by the differential localization of three histidine kinases (PleC, CckA and DivJ) and the response regulators that they control (CtrA, DivK and PleD). PleC and CckA localize predominantly to the nascent flagellar pole 33,65, whereas DivJ is found mostly at the stalked pole 65. PleC has activity as a DivK~P phosphatase and acts to inhibit, perhaps as a phosphatase, the formation of phosphorylated PleD (PleD~P) in vivo 37,65,66. By contrast, DivJ acts as a DivK and PleD kinase 37,66. The combination of these factors leads to higher concentrations of PleD~P and DivK~P in stalked cells than in swarmer cells. PleD~P is required for loss of flagellar motility and DivK~P is required for CtrA degradation; therefore, higher concentrations of these two active response regulators in stalked cells helps determine key aspects of stalked cell fate. CckA is required for CtrA phosphorylation and is predominantly localized to the swarmer pole. This presumably helps establish active CtrA only in the swarmer cell where it can inhibit DNA replication. tetratricopeptide repeat motifs (TPR), which are motifs that are known to mediate protein protein interactions in a number of other systems 74. How is PodJ localized to the cell pole? Intriguingly, the extreme C-terminus of PodJ has weak homology to peptidoglycan-binding domains. So, one possibility is that PodJ interacts with a cell wall structure that is itself specifically located at the nascent swarmer pole, which, as the site of cell division in the preceding cell cycle, might contain a peptidoglycan structure that was formerly associated with septum assembly. Although little is known about how PodJ functions, this protein clearly has an important role in the spatial patterning processes in Caulobacter, and it will prove fascinating as the mechanisms of localization are unravelled. Homologues of PodJ are found throughout the α-subdivision of proteobacteria, so PodJ could have an important role in establishing cell polarity in many bacteria. Future directions Caulobacter has become the predominant model system for understanding the regulation of bacterial cellcycle progression at a molecular level. Studying the Caulobacter cell-division cycle has begun to reveal the mechanisms by which bacterial cells establish and maintain spatial asymmetry. Specifically, two-component signal transduction proteins have emerged as factors that are of prime importance in the control of the cell cycle and asymmetric cell division. The advent and application of genomic technologies has revolutionized the pace of progress in mapping and understanding the intricate genetic network controlling the Caulobacter life cycle. Global analyses have so far focused on transcriptional regulation, because DNA microarrays, the most mature of genomic technologies, are designed to assay gene expression and RNA levels. Global analyses of other modes of regulation, including post-translational modifications, protein protein interactions and subcellular localization, will be important in the development of a more complete, molecular-level picture of cell-cycle progression in Caulobacter. Better computational tools will be needed to assemble, store, analyse and mine these new and complex data sets. In this era of global analyses, new challenges also arise beyond understanding the role of individual components in the cell-cycle regulatory network. For example, how does a single Caulobacter cell coordinate the activities of more than 100 two-component signalling proteins? Given that these proteins are very closely related to each other, how does the cell ensure specificity of cognate pairs and prevent unwanted cross-talk? In the case of response regulators such as DivK and CtrA, which are controlled by numerous kinases, is there signal integration? Or are these pathways responding to different signals at different times? Systematic analysis of the two-component systems in Caulobacter will be needed to begin addressing these systems-level questions. We anticipate that such analyses will illuminate general design principles and regulatory schemes that are used to ensure the fidelity of cell-cycle progression and to generate cellular asymmetry. NATURE REVIEWS MICROBIOLOGY VOLUME 2 APRIL