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Regulatory circuits in Caulobacter
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Regulatory circuits in Caulobacter Magne Østerås and Urs Jenal* The transcriptional regulator CtrA controls DNA replication, DNA methylation and cell division during the Caulobacter cell cycle. Recent work on this master cell cycle regulator has provided insight into its control mechanisms. Feedback regulation of ctrA transcription together with timed proteolysis of CtrA determine the levels of the regulator during the cell cycle. Multiple phosphorylation pathways regulate CtrA activity by a mechanism that includes dynamic subcellular localization of the corresponding sensor kinases. Addresses Division of Molecular Microbiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland *e-mail: [email protected] Current Opinion in Microbiology 2000, 3:171–176 1369-5274/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviation Cori chromosomal origin of replication
Introduction The life cycle of Caulobacter crescentus includes an asymmetric cell division. The two compartments of each dividing cell acquire different developmental programs resulting in a sessile stalked cell and a motile swarmer cell progeny (Figure 1). The stalked cell immediately initiates a new round of chromosomal DNA replication and cell division, whereas the swarmer cell must first differentiate into a stalked cell to be able to start a new replicative cycle. A single polar flagellum coupled to a chemosensory signal Figure 1 Schematic diagram of the Caulobacter crescentus cell cycle. The major features of the different cell types and the stages of the cell cycle (G1, S, G2) are indicated. The presynthetic gap (G1) corresponds to the motile, replication silent swarmer cell. The period of active DNA synthesis (S) includes the stalked and early predivisional cell. The postsynthetic gap (G2) starts in the late predivisional cell when replication is completed and lasts until cell division has occurred. The timing of cell cycle-related developmental events is indicated and polar appendices are marked.
transduction apparatus allows the non-replicating swarmer cell to perform chemotaxis in liquid environments. This motile period (G1 phase) is followed by the loss of the flagellum and the chemotaxis apparatus during the swarmer-to-stalked cell transition, and the synthesis of a stalk at the pole previously occupied by the flagellum. This morphological transition coincides with the initiation of DNA replication (S phase). The stalked cell elongates during S phase and assembles a new flagellum at the pole opposite of the stalk (Figure 1). Upon completion of DNA replication the chromosomes move to the poles, followed by cytokinesis and cell division (G2 phase). Each Caulobacter division cycle is thus characterized by a transition between a motile, replication silent cell type and a sessile, replication competent cell. This poses some important questions. What regulatory circuits and checkpoints determine the order and periodicity of cell cycle events in Caulobacter? How is cell differentiation integrated into such a regulatory network? Here we review the recent progress made towards understanding the temporal and spatial regulation during the C. crescentus cell cycle. The new findings emphasize that in order to fully appreciate the complexity of regulatory circuits in bacteria, one has to consider changes in protein expression, activity and turnover, as well as dynamic rearrangements of the subcellular organization.
CtrA, a master cell cycle regulator The ctrA gene was isolated in experiments designed to genetically identify regulatory components that link the expression of flagellar genes to the Caulobacter cell cycle [1].
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Figure 2 Model depicting the proposed mechanisms used to control CtrA levels and activity and the role of phosphorylated CtrA (CtrA~P) in transcriptional regulation. The sensor kinases DivJ, DivL and CckA are activated in response to as yet unknown cell cycle cues (?). The phosphoryl group is transferred to CtrA either directly or via a multicomponent phosphorelay. The different promoters that are regulated positively or negatively by CtrA~P are indicated and discussed further in the text. The CtrA-binding site (CtrA box) is shown schematically. The ctrA promoters P1 and P2 are indicated. Positive autoregulation of P2 and negative autoregulation of P1 contribute to the temporal expression profile of CtrA during the cell cycle (Figure 3). Degradation of CtrA by the ClpXP protease is indicated. No evidence exists for selective degradation of either CtrA or CtrA~P.
CtrA is a member of the response-regulator family of transcriptional regulators (for more information on response regulators see the review by Hoch, this issue pp 165–170) and is used by the cell to control both developmental and cell cycle events. In vivo and in vitro experiments have demonstrated that CtrA, by activating transcription of early flagellar genes, starts the process of flagellar assembly in the predivisional cell [1,2••,3•]. In addition, CtrA is involved in the control of several essential cell cycle processes including DNA methylation, cell division and DNA replication (Figure 2). CtrA is required for the transcription of the ccrM gene, which encodes a DNA methyltransferase essential for C. crescentus growth [1,3•,4]. Transcription of ccrM is restricted to the G2 phase of the cell cycle [5] (Figure 3). Because the CcrM protein is also rapidly degraded by the Lon protease, CcrM is only present when it is actively synthesized. As a result, the newly replicated chromosomes persist in a hemimethylated state for an extended period of the cell cycle, a feature that seems to be used by the cell to avoid premature reinitiation of DNA replication [6]. CtrA is also a timing device for the expression of cell division genes. CtrA represses transcription of the ftsZ gene in swarmer cells [7•]. Transcription of the ftsQA operon, however, is induced by CtrA in predivisional cells (Y Brun, unpublished data). Thus, CtrA contributes to the sequential transcription of ftsZ and ftsQA during the cell cycle (Figure 3) [8•]. This sequence of gene expression conforms to the order of assembly of bacterial cytokinesis components at mid-cell [9]. Most importantly, CtrA negatively regulates the activity of a strong promoter (PCori) located inside the Caulobacter chromosomal origin of replication (Cori) [1,10••]. PCori activity is strictly required for the initiation of DNA
replication [11]. CtrA binds to a conserved DNA-sequence motif (the CtrA box), which is found in all CtrA-controlled promoters [1,3•,7•,10••] (Figure 2). The CtrA box overlaps with the –35 region of promoters that are activated by CtrA, whereas it is located close to the transcriptional start site of promoters that are negatively controlled by CtrA. The minimal Cori region contains five CtrA boxes, two of which overlap with the PCori region. CtrA represses PCori activity in all cell types except in the stalked cell in which initiation of DNA replication takes place [10••] (Figure 3). The crucial role of CtrA in controlling replication initiation is supported by the observation that reducing CtrA activity in vivo causes over-replication and the accumulation of multiple chromosomes per cell, whereas increasing CtrA activity results in a replication block [10••,12].
Regulation of CtrA levels by timed synthesis and degradation How can a single regulatory component execute all these different functions at distinct stages of the cell cycle? It is not surprising to find that the cell has imposed tight temporal and spatial control upon CtrA activity. Just as with most other response regulators, CtrA is only active when it is phosphorylated. However, active CtrA is restricted to the predivisional and swarmer cell types (Figure 3). This is accomplished by at least partially redundant control mechanisms at the level of CtrA expression, stability, and phosphorylation [12]. CtrA synthesis itself is under cell cycle control, in that transcription of the ctrA gene only occurs in the predivisional cell after DNA replication has been initiated (Figure 3). Two distinct promoters contribute to ctrA transcription: a weaker promoter (P1) that is active only in the early predivisional cell, and a stronger promoter (P2) that is active in the late predivisional cell. The activity of P1 is repressed by CtrA itself, whereas P2 is activated by CtrA (Figure 2)
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[13•]. Thus, as levels of phosphorylated CtrA build up in the early predivisional cell, P1 is eventually turned off, while P2 is switched on to full activity resulting in high CtrA concentrations at the end of S phase. This simple autoregulatory mechanism provides the basis for the correct timing of the periodic peaks of CtrA expression. ‘Resetting the clock’ requires the removal of CtrA at the proper time in the cell cycle. This would result in P1 activation to restart the cycle.
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Figure 3
CtrA is indeed proteolytically degraded in the stalked compartment of the late predivisional cell and during the swarmer-to-stalked cell transition (Figure 3) [12]. CtrA degradation control can override defects in transcriptional control [12], emphasizing its important role for the periodic fluctuations of CtrA levels. Changing levels of CtrA in the predivisional cell may also explain variations in the temporal control of CtrA-dependent promoters. CtrA has a 20-fold higher affinity for the flagellar fliQ promoter compared with its affinity for the ccrM promoter [3•]. While fliQ and other CtrA-dependent flagellar promoters are transcribed early in the predivisional cell when CtrA levels are still low, ccrM expression is only activated late in the cell cycle after CtrA levels have built up (Figure 3). Removal of active CtrA before cells enter S phase is not only important for CtrA autoregulation but is also crucial for the activation of the PCori promoter and replication initiation (Figure 3). Mutations in ctrA that result in a constitutively active and stable CtrA protein result in a G1 cell cycle arrest [12]. Degradation of CtrA requires the ATP-dependent ClpXP protease [14•,15]. Inactivation of clpP or clpX is lethal and also results in a G1 arrest [14•]. However, mutant ctrA alleles causing a stable CtrA protein are viable as long as normal phosphorylation control is maintained [12]. Thus, the requirement for CtrA turnover alone does not explain the essential nature of the ClpXP protease in C. crescentus [14•]. But how is the CtrA proteolytic activity turned on and off during the cell cycle? Since the levels of ClpXP do not change during the cell cycle [14•], additional proteins must exist which control either the ClpXP proteolytic activity or the accessibility of CtrA. Although precedence for such post-translational control of transcription factors exists in Escherichia coli and Bacillus subtilis [16–19], the molecular basis for ClpXP-mediated degradation of CtrA remains to be elucidated.
Regulation of CtrA activity by multiple phosphorylation pathways In general, response-regulator proteins are activated through phosphorylation by sensor kinases in response to environmental signals [20]. Newly synthesized CtrA is immediately phosphorylated and no less than three kinases — DivJ, DivL and CckA — seem to be involved in this process (Figure 2) [2••,21••,22••]. Even though the signals recognized by these kinases are not known, two of them, DivJ and CckA, localize to distinct subcellular sites, indicating that spatial cues could contribute to the activation of
Distribution of CtrA and subcellular localization of the sensor kinases CckA, PleC and DivJ during the cell cycle. Periods of CtrA synthesis and degradation during the cell cycle are outlined in the top panel. Cellular levels of CtrA are also indicated in the top panel as different shades of gray: no CtrA (white); low CtrA concentration (light gray); high CtrA concentration (dark gray). The time periods of activity of the CtrA-regulated promoters discussed in the text are outlined by horizontal boxes. The three lower panels show the subcellular distribution of the CckA, PleC and DivJ kinases. CckA and PleC are present in the membrane throughout the cell cycle. CckA moves to the swarmer pole in the early predivisional cell and delocalizes in the late predivisional cell before division. PleC localizes to the swarmer pole of the late predivisional cell, when flagellar assembly is completed, and remains at this pole for the entire motile phase of the cell cycle. Coincident with cell differentiation, PleC delocalizes and is replaced by DivJ, which marks the stalked pole.
CtrA (see below). Biochemical and genetic evidence has proposed a role for the histidine kinase DivJ and for the essential single domain response regulator DivK in CtrA phosphorylation [2••,23,24]. DivJ is able to efficiently phosphorylate DivK but not CtrA in vitro [2••] and it contributes substantially to DivK phosphorylation in vivo
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[25••]. In addition, a mutant ctrA allele (sokA) suppresses the lethality phenotype of a divK null mutation [2••]. These data postulate the presence of a phosphorelay [20] with the phosphoryl group transferred from DivJ to the phosphotransfer protein DivK and from there via a still unknown component to CtrA (Figure 2). The sokA allele can also bypass the conditional cell division phenotype of cold-sensitive divL mutants [21••]. The DivL kinase is essential for cell viability and instead of being phosphorylated on a histidine residue, autophosphorylates on a tyrosine residue [21••]. DivL can catalyze CtrA phosphorylation and activation in vitro, suggesting that this kinase controls CtrA via a DivJ/DivK-independent pathway (Figure 2). A third signal transduction pathway leading to the activation of CtrA is defined by the CckA histidine kinase (Figure 2). CckA is essential for C. crescentus growth and is required for CtrA phosphorylation in vivo [22••]. CckA belongs to the group of hybrid kinases that contain both a kinase and a receiver domain of response regulators [26]. The CckA receiver domain could be involved in phosphotransfer from the kinase domain to an as yet unidentified phosphotransferase as the link between CckA and CtrA. Alternatively, the phosphoryl group could be transferred directly from CckA to CtrA with the CckA receiver domain playing a regulatory role in this process. It is interesting to note that out of 51 different histidine kinases encoded by the C. crescentus chromosome at least 20 are hybrid kinases. This compares with five hybrid kinases among 29 reported histidine kinases in E. coli [27]. It can be anticipated that the complexity of this regulatory network will increase and that additional signal transduction components such as phosphotransfer proteins or phospho-protein phosphatases will be found to regulate CtrA activity. But why are multiple non-redundant pathways required for the regulation of one common response regulator? It is likely that this regulatory network has to integrate a multitude of different structural and physiological signals before it ‘allows’ CtrA to make a number of very vital ‘decisions’ during cell propagation.
Differential localization of the CtrA sensor kinases The most fascinating aspect of the CtrA signal transduction pathway is provided by the recent observation that several of the sensor kinases undergo dynamic rearrangement during the cell cycle and localize to specific subcellular sites. Just as with most sensor protein kinases, CckA is membrane bound and evenly distributed throughout most of the cell cycle. However, in the early predivisional cell, coinciding with the onset of CtrA phosphorylation, CckA concentrates at the pole opposite the stalk [22••] (Figure 3). Polar positioning of CckA is maintained until immediately before the cells divide. Initial evidence suggests that polar localization is required for CckA activity and phosphorylation of CtrA [22••]. The histidine kinase PleC is distributed evenly in the membrane
of stalked and early predivisional cells before it dynamically rearranges to the pole that carries the flagellum and the chemosensory proteins [25••] (Figure 3). During the G1 to S transition, PleC delocalizes and the DivJ kinase concentrates at the stalked pole (Figure 3). PleC controls polar morphogenesis and motility [28], and is required for polar localization and possibly the activation of DivJ [25••]. The finding that these sensory components specifically localize to the Caulobacter cell poles at distinct stages of the cell cycle suggests that CtrA activity is controlled at least in part by the nature of the poles. Every newly formed pole at cell division develops into a flagellated swarmer pole and then into a stalked pole. The fact that poles are ‘marked’ through development by successive positioning of different kinases, together with the finding that positioning of a given kinase might be dependent on the activity or positioning of the preceding kinase, suggests that the different phosphorylation pathways, rather than acting on CtrA in parallel, are in fact interdependent pathways of an intricate regulatory network.
Concluding remarks: are checkpoints involved in cell cycle control? Although the mechanisms and components used to control CtrA levels and activity are currently being unraveled, the nature of the cell cycle signals that elicit the CtrA response remain unknown. As an attractive model one can postulate checkpoints that would time and coordinate different events as the cell cycle proceeds. But is there evidence for checkpoint control in Caulobacter? An ideal timing device would be the methylation status of the chromosome. Disrupting the normal cell cycle methylation pattern has indeed drastic consequences for cell growth and replication control [4,6]. In addition, there is evidence that CcrM-dependent DNA methylation contributes to the cell cycle transcription profile of the ccrM gene [5,29]. Thus, DNA methylation acts as a timing device in the Caulobacter cell cycle and additional events might exist that rely on this signal for correct temporal control. DNA replication could also act as cell cycle checkpoint. Inhibition of DNA replication in C. crescentus leads to a rapid loss of the promoter activity of early flagellar genes and the ccrM gene [5,30,31]. Because all of these promoters are controlled by CtrA, CtrA activity itself might be dependent on active DNA replication. Thus, CtrA could control replication, and in turn be controlled by replication in a feedback process. Recently, chromosome segregation has been added to the list of possible cell cycle checkpoints. The Caulobacter Cori regions are dynamically positioned to the cell poles immediately after the initiation of DNA replication [32•]. Proteins that are involved in this process include ParA, ParB and SMC [32•,33]. ParB binds to a region of the chromosome close to the origin and, together with ParA, was proposed to be involved in directing the movement of the DNA to the cell poles [33]. A null mutation in the smc gene is
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conditionally lethal and at the permissive conditions causes mislocalized origins and abnormal nucleoid morphology. When grown under restrictive conditions, the smc mutant arrests as predivisional cells with two fully replicated chromosomes [32•]. Interestingly, the blocked mutant cells fail to build up CtrA levels, suggesting that either CtrA synthesis is not turned on or CtrA degradation never turned off under these conditions [32•]. Thus, the correct structure and/or positioning of the chromosome could act as a checkpoint early in the S phase and in response trigger the increase of CtrA levels in the predivisional cell. One possibility is that the movement of proteins associated with the origin of replication to the poles constitutes a localized signal for later steps in the cell cycle. This brings us right back to the cell poles. Future work will have to be directed towards understanding the role of the poles as part of the regulatory network controlling Caulobacter cell cycle and development.
Acknowledgements The authors thank P Aldridge, C Stephens and RT Wheeler for critically reading the manuscript. We thank Y Brun for communicating results prior to publication. U Jenal is supported by the Swiss National Science Foundation grant 31-46764.96.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Quon KC, Marczynski GT, Shapiro L: Cell cycle control by an essential bacterial two-component signal transduction protein. Cell 1996, 84:83-93.
2. ••
Wu J, Ohta N, Newton A: An essential, multicomponent signal transduction pathway required for cell cycle regulation in Caulobacter. Proc Natl Acad Sci USA 1998, 95:1443-1448. The authors present genetic evidence that the essential single-domain response regulator DivK is involved in CtrA activation. A biochemical analysis demonstrates that the sensor kinase DivJ phosphorylates DivK preferentially over CtrA. The authors also show that the phosphorylated form of CtrA is required for in vitro transcription of a flagellar gene. This work postulates that a multicomponent phosphorelay is involved in CtrA activation in Caulobacter. 3. •
Reisenauer A, Quon K, Shapiro L: The CtrA response regulator mediates temporal control of gene expression during the Caulobacter cell cycle. J Bacteriol 1999, 181:2430-2439. The authors show that the differential temporal control of CtrA-regulated genes during the cell cycle can in part be explained by the different affinity of phosphorylated CtrA for their promoters, thus correlating the timing of transcription with the cellular levels of CtrA. 4.
Stephens C, Reisenauer A, Wright R, Shapiro L: A cell cycleregulated bacterial DNA methyltransferase is essential for viability. Proc Natl Acad Sci USA 1996, 93:1210-1214.
5.
Stephens CM, Zweiger G, Shapiro L: Coordinate cell cycle control of a Caulobacter DNA methyltransferase and the flagellar genetic hierarchy. J Bacteriol 1995, 177:1662-1669.
6.
Wright R, Stephens C, Zweiger G, Shapiro L, Alley MR: Caulobacter Lon protease has a critical role in cell-cycle control of DNA methylation. Genes Dev 1996, 10:1532-1542.
7. •
Kelly AJ, Sackett MJ, Din N, Quardokus E, Brun YV: Cell cycle dependent transcriptional and proteolytic regulation of FtsZ in Caulobacter. Genes Dev 1998, 12:880-893. This work provides the regulatory basis for the observation that the cytokinesis protein FtsZ is not present in non-replicating swarmer cells. The cell cycle control of FtsZ includes CtrA-regulated expression and cell type-specific proteolysis. Newly synthesized FtsZ is stable when it is assembled at mid-cell at the beginning of the cell cycle. FtsZ degradation seems to be coupled to its disassembly after cell division.
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Sackett MJ, Kelly AJ, Brun YV: Ordered expression of ftsQA and ftsZ during the Caulobacter crescentus cell cycle. Mol Microbiol 1998, 28:421-434. This paper demonstrates that the ftsQA genes are transcribed independently of and in sequence with ftsZ during the cell cycle. 9.
Rothfield LI, Justice SS: Bacterial cell division: the cycle of the ring. Cell 1997, 88:581-584.
10. Quon KC, Yang B, Domian IJ, Shapiro L, Marczynski GT: Negative •• control of bacterial DNA replication by a cell cycle regulatory protein that binds at the chromosome origin. Proc Natl Acad Sci USA 1998, 95:120-125. The authors describe the mechanisms of DNA replication control by CtrA. CtrA represses the initiation of DNA replication by binding to five CtrA boxes inside the origin of replication. This restricts replication initiation to the phase of the cell cycle when CtrA is not present in the cell. 11. Marczynski GT, Lentine K, Shapiro L: A developmentally regulated chromosomal origin of replication uses essential transcription elements. Genes Dev 1995, 9:1543-1557. 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. Domian IJ, Reisenauer A, Shapiro L: Feedback control of a master • bacterial cell-cycle regulator. Proc Natl Acad Sci USA 1999, 96:6648-6653. This work explains the mechanisms of ctrA transcriptional autoregulation. Two ctrA promoters are active in the early and late predivisional cells, respectively. The first is repressed by CtrA and thus activated after the initiation of DNA replication when the regulator is absent. The accumulation of CtrA will then activate the second promoter resulting in high CtrA levels in late predivisional cells. 14. Jenal U, Fuchs T: An essential protease involved in bacterial cell • cycle control. EMBO J 1998, 17:5658-5669. The authors demonstrate that the Caulobacter ClpXP protease is required for cell cycle progression and for CtrA degradation during the G1 to S transition. 15. Østerås M, Stotz A, Schmid Nuoffer S, Jenal U: Identification and transcriptional control of the genes encoding the Caulobacter crescentus ClpXP protease. J Bacteriol 1999, 181:3039-3050. 16. Zhou Y, Gottesman S: Regulation of proteolysis of the stationaryphase sigma factor RpoS. J Bacteriol 1998, 180:1154-1158. 17.
Tatsuta T, Tomoyasu T, Bukau B, Kitagawa M, Mori H, Karata K, Ogura T: Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of sigma32 in vivo. Mol Microbiol 1998, 30:583-593.
18. Turgay K, Hahn J, Burghoorn J, Dubnau D: Competence in Bacillus subtilis is controlled by regulated proteolysis of a transcription factor. EMBO J 1998, 17:6730-6738. 19. Becker G, Klauck E, Hengge-Aronis R: Regulation of RpoS proteolysis in Escherichia coli: the response regulator RssB is a recognition factor that interacts with the turnover element in RpoS. Proc Natl Acad Sci USA 1999, 96:6439-6444. 20. Perraud AL, Weiss V, Gross R: Signalling pathways in twocomponent phosphorelay systems. Trends Microbiol 1999, 7:115-120. 21. Wu J, Ohta N, Zhao J, Newton A: A novel bacterial tyrosine kinase •• essential for cell division and differentiation. Proc Natl Acad Sci USA 1999, 96:13068-13073. This paper shows that the sensor kinase, DivL, which autophosphorylates on a tyrosine instead of the usual histidine residue, can phosphorylate CtrA in vitro. The divL gene is essential for viability of Caulobacter. The lethal phenotype of a divL mutant can be suppressed by a mutant allele of ctrA linking the DivL sensor to the CtrA regulatory network. 22. Jacobs C, Domian IJ, Maddock JR, Shapiro L: Cell cycle-dependent •• polar localization of an essential bacterial histidine kinase that controls DNA replication and cell division. Cell 1999, 97:111-120. This paper describes the identification of CckA, a sensor kinase required for phosphorylation of CtrA in vivo. CckA is a membrane-bound protein that localizes to the swarmer pole in the early predivisional cell, but is evenly distributed during the rest of the cell cycle. Evidence is presented suggesting that correct localization of CckA is important for its activity. 23. 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.
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24. Ohta N, Lane T, Ninfa EG, Sommer JM, Newton A: A histidine protein kinase homologue required for regulation of bacterial cell division and differentiation. Proc Natl Acad Sci USA 1992, 89:10297-10301. 25. Wheeler R, Shapiro L: Differential localization of two histidine •• kinases controlling bacterial cell differentiation. Mol Cell 1999, 4:683-694. The two sensor kinases, PleC and DivJ, show a distinct pattern of subcellular localization during the cell cycle. PleC is localized to the swarmer pole of late predivisional cells and is replaced at this site by DivJ during the swarmer to stalk transition. Interestingly, correct localization of DivJ requires PleC. 26. Appleby JL, Parkinson JS, Bourret RB: Signal transduction via the multi-step phosphorelay: not necessarily a road less traveled. Cell 1996, 86:845-848. 27.
Ohta N, Grebe TW, Newton A: Signal transduction and cell cycle checkpoints in developmental regulation of Caulobacter. In Prokaryotic Development. Edited by Brun YV, Shimkets LJ. Washington, DC: ASM Press; 1999:341-359.
28. Wang SP, Sharma PL, Schoenlein PV, Ely B: A histidine protein kinase is involved in polar organelle development in Caulobacter crescentus. Proc Natl Acad Sci USA 1993, 90:630-634.
29. Reisenauer A, Kahng LS, McCollum S, Shapiro L: Bacterial DNA methylation: a cell cycle regulator? J Bacteriol 1999, 181:51355139. 30. Dingwall A, Zhuang WY, Quon K, Shapiro L: Expression of an early gene in the flagellar regulatory hierarchy is sensitive to an interruption in DNA replication. J Bacteriol 1992, 174:1760-1768. 31. Stephens CM, Shapiro L: An unusual promoter controls cell-cycle regulation and dependence on DNA replication of the Caulobacter fliLM early flagellar operon. Mol Microbiol 1993, 9:1169-1179. 32. Jensen RB, Shapiro L: The Caulobacter crescentus smc gene is • required for cell cycle progression and chromosome segregation. Proc Natl Acad Sci USA 1999, 96:10661-10666. The authors used fluorescence in situ hybridization to show that the Caulobacter origins of replication move to opposite cell poles immediately after initiation of replication. A conditionally lethal smc null mutant results in abnormal DNA positioning and cell cycle arrest at the predivisional stage. Arrested smc mutants complete DNA replication but fail to build up CtrA levels. This observation proposes a segregation checkpoint upstream of CtrA activity in the predivisional cell. 33. Mohl DA, Gober JW: Cell cycle-dependent polar localization of chromosome partitioning proteins in Caulobacter crescentus. Cell 1997, 88:675-684.
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Regulatory circuits in Caulobacter Magne Østerås and Urs Jenal* The transcriptional regulator CtrA controls DNA replication, DNA methylation and cell division during the Caulobacter cell cycle. Recent work on this master cell cycle regulator has provided insight into its control mechanisms. Feedback regulation of ctrA transcription together with timed proteolysis of CtrA determine the levels of the regulator during the cell cycle. Multiple phosphorylation pathways regulate CtrA activity by a mechanism that includes dynamic subcellular localization of the corresponding sensor kinases. Addresses Division of Molecular Microbiology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland *e-mail: [email protected] Current Opinion in Microbiology 2000, 3:171–176 1369-5274/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviation Cori chromosomal origin of replication
Introduction The life cycle of Caulobacter crescentus includes an asymmetric cell division. The two compartments of each dividing cell acquire different developmental programs resulting in a sessile stalked cell and a motile swarmer cell progeny (Figure 1). The stalked cell immediately initiates a new round of chromosomal DNA replication and cell division, whereas the swarmer cell must first differentiate into a stalked cell to be able to start a new replicative cycle. A single polar flagellum coupled to a chemosensory signal Figure 1 Schematic diagram of the Caulobacter crescentus cell cycle. The major features of the different cell types and the stages of the cell cycle (G1, S, G2) are indicated. The presynthetic gap (G1) corresponds to the motile, replication silent swarmer cell. The period of active DNA synthesis (S) includes the stalked and early predivisional cell. The postsynthetic gap (G2) starts in the late predivisional cell when replication is completed and lasts until cell division has occurred. The timing of cell cycle-related developmental events is indicated and polar appendices are marked.
transduction apparatus allows the non-replicating swarmer cell to perform chemotaxis in liquid environments. This motile period (G1 phase) is followed by the loss of the flagellum and the chemotaxis apparatus during the swarmer-to-stalked cell transition, and the synthesis of a stalk at the pole previously occupied by the flagellum. This morphological transition coincides with the initiation of DNA replication (S phase). The stalked cell elongates during S phase and assembles a new flagellum at the pole opposite of the stalk (Figure 1). Upon completion of DNA replication the chromosomes move to the poles, followed by cytokinesis and cell division (G2 phase). Each Caulobacter division cycle is thus characterized by a transition between a motile, replication silent cell type and a sessile, replication competent cell. This poses some important questions. What regulatory circuits and checkpoints determine the order and periodicity of cell cycle events in Caulobacter? How is cell differentiation integrated into such a regulatory network? Here we review the recent progress made towards understanding the temporal and spatial regulation during the C. crescentus cell cycle. The new findings emphasize that in order to fully appreciate the complexity of regulatory circuits in bacteria, one has to consider changes in protein expression, activity and turnover, as well as dynamic rearrangements of the subcellular organization.
CtrA, a master cell cycle regulator The ctrA gene was isolated in experiments designed to genetically identify regulatory components that link the expression of flagellar genes to the Caulobacter cell cycle [1].
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Figure 2 Model depicting the proposed mechanisms used to control CtrA levels and activity and the role of phosphorylated CtrA (CtrA~P) in transcriptional regulation. The sensor kinases DivJ, DivL and CckA are activated in response to as yet unknown cell cycle cues (?). The phosphoryl group is transferred to CtrA either directly or via a multicomponent phosphorelay. The different promoters that are regulated positively or negatively by CtrA~P are indicated and discussed further in the text. The CtrA-binding site (CtrA box) is shown schematically. The ctrA promoters P1 and P2 are indicated. Positive autoregulation of P2 and negative autoregulation of P1 contribute to the temporal expression profile of CtrA during the cell cycle (Figure 3). Degradation of CtrA by the ClpXP protease is indicated. No evidence exists for selective degradation of either CtrA or CtrA~P.
CtrA is a member of the response-regulator family of transcriptional regulators (for more information on response regulators see the review by Hoch, this issue pp 165–170) and is used by the cell to control both developmental and cell cycle events. In vivo and in vitro experiments have demonstrated that CtrA, by activating transcription of early flagellar genes, starts the process of flagellar assembly in the predivisional cell [1,2••,3•]. In addition, CtrA is involved in the control of several essential cell cycle processes including DNA methylation, cell division and DNA replication (Figure 2). CtrA is required for the transcription of the ccrM gene, which encodes a DNA methyltransferase essential for C. crescentus growth [1,3•,4]. Transcription of ccrM is restricted to the G2 phase of the cell cycle [5] (Figure 3). Because the CcrM protein is also rapidly degraded by the Lon protease, CcrM is only present when it is actively synthesized. As a result, the newly replicated chromosomes persist in a hemimethylated state for an extended period of the cell cycle, a feature that seems to be used by the cell to avoid premature reinitiation of DNA replication [6]. CtrA is also a timing device for the expression of cell division genes. CtrA represses transcription of the ftsZ gene in swarmer cells [7•]. Transcription of the ftsQA operon, however, is induced by CtrA in predivisional cells (Y Brun, unpublished data). Thus, CtrA contributes to the sequential transcription of ftsZ and ftsQA during the cell cycle (Figure 3) [8•]. This sequence of gene expression conforms to the order of assembly of bacterial cytokinesis components at mid-cell [9]. Most importantly, CtrA negatively regulates the activity of a strong promoter (PCori) located inside the Caulobacter chromosomal origin of replication (Cori) [1,10••]. PCori activity is strictly required for the initiation of DNA
replication [11]. CtrA binds to a conserved DNA-sequence motif (the CtrA box), which is found in all CtrA-controlled promoters [1,3•,7•,10••] (Figure 2). The CtrA box overlaps with the –35 region of promoters that are activated by CtrA, whereas it is located close to the transcriptional start site of promoters that are negatively controlled by CtrA. The minimal Cori region contains five CtrA boxes, two of which overlap with the PCori region. CtrA represses PCori activity in all cell types except in the stalked cell in which initiation of DNA replication takes place [10••] (Figure 3). The crucial role of CtrA in controlling replication initiation is supported by the observation that reducing CtrA activity in vivo causes over-replication and the accumulation of multiple chromosomes per cell, whereas increasing CtrA activity results in a replication block [10••,12].
Regulation of CtrA levels by timed synthesis and degradation How can a single regulatory component execute all these different functions at distinct stages of the cell cycle? It is not surprising to find that the cell has imposed tight temporal and spatial control upon CtrA activity. Just as with most other response regulators, CtrA is only active when it is phosphorylated. However, active CtrA is restricted to the predivisional and swarmer cell types (Figure 3). This is accomplished by at least partially redundant control mechanisms at the level of CtrA expression, stability, and phosphorylation [12]. CtrA synthesis itself is under cell cycle control, in that transcription of the ctrA gene only occurs in the predivisional cell after DNA replication has been initiated (Figure 3). Two distinct promoters contribute to ctrA transcription: a weaker promoter (P1) that is active only in the early predivisional cell, and a stronger promoter (P2) that is active in the late predivisional cell. The activity of P1 is repressed by CtrA itself, whereas P2 is activated by CtrA (Figure 2)
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[13•]. Thus, as levels of phosphorylated CtrA build up in the early predivisional cell, P1 is eventually turned off, while P2 is switched on to full activity resulting in high CtrA concentrations at the end of S phase. This simple autoregulatory mechanism provides the basis for the correct timing of the periodic peaks of CtrA expression. ‘Resetting the clock’ requires the removal of CtrA at the proper time in the cell cycle. This would result in P1 activation to restart the cycle.
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Figure 3
CtrA is indeed proteolytically degraded in the stalked compartment of the late predivisional cell and during the swarmer-to-stalked cell transition (Figure 3) [12]. CtrA degradation control can override defects in transcriptional control [12], emphasizing its important role for the periodic fluctuations of CtrA levels. Changing levels of CtrA in the predivisional cell may also explain variations in the temporal control of CtrA-dependent promoters. CtrA has a 20-fold higher affinity for the flagellar fliQ promoter compared with its affinity for the ccrM promoter [3•]. While fliQ and other CtrA-dependent flagellar promoters are transcribed early in the predivisional cell when CtrA levels are still low, ccrM expression is only activated late in the cell cycle after CtrA levels have built up (Figure 3). Removal of active CtrA before cells enter S phase is not only important for CtrA autoregulation but is also crucial for the activation of the PCori promoter and replication initiation (Figure 3). Mutations in ctrA that result in a constitutively active and stable CtrA protein result in a G1 cell cycle arrest [12]. Degradation of CtrA requires the ATP-dependent ClpXP protease [14•,15]. Inactivation of clpP or clpX is lethal and also results in a G1 arrest [14•]. However, mutant ctrA alleles causing a stable CtrA protein are viable as long as normal phosphorylation control is maintained [12]. Thus, the requirement for CtrA turnover alone does not explain the essential nature of the ClpXP protease in C. crescentus [14•]. But how is the CtrA proteolytic activity turned on and off during the cell cycle? Since the levels of ClpXP do not change during the cell cycle [14•], additional proteins must exist which control either the ClpXP proteolytic activity or the accessibility of CtrA. Although precedence for such post-translational control of transcription factors exists in Escherichia coli and Bacillus subtilis [16–19], the molecular basis for ClpXP-mediated degradation of CtrA remains to be elucidated.
Regulation of CtrA activity by multiple phosphorylation pathways In general, response-regulator proteins are activated through phosphorylation by sensor kinases in response to environmental signals [20]. Newly synthesized CtrA is immediately phosphorylated and no less than three kinases — DivJ, DivL and CckA — seem to be involved in this process (Figure 2) [2••,21••,22••]. Even though the signals recognized by these kinases are not known, two of them, DivJ and CckA, localize to distinct subcellular sites, indicating that spatial cues could contribute to the activation of
Distribution of CtrA and subcellular localization of the sensor kinases CckA, PleC and DivJ during the cell cycle. Periods of CtrA synthesis and degradation during the cell cycle are outlined in the top panel. Cellular levels of CtrA are also indicated in the top panel as different shades of gray: no CtrA (white); low CtrA concentration (light gray); high CtrA concentration (dark gray). The time periods of activity of the CtrA-regulated promoters discussed in the text are outlined by horizontal boxes. The three lower panels show the subcellular distribution of the CckA, PleC and DivJ kinases. CckA and PleC are present in the membrane throughout the cell cycle. CckA moves to the swarmer pole in the early predivisional cell and delocalizes in the late predivisional cell before division. PleC localizes to the swarmer pole of the late predivisional cell, when flagellar assembly is completed, and remains at this pole for the entire motile phase of the cell cycle. Coincident with cell differentiation, PleC delocalizes and is replaced by DivJ, which marks the stalked pole.
CtrA (see below). Biochemical and genetic evidence has proposed a role for the histidine kinase DivJ and for the essential single domain response regulator DivK in CtrA phosphorylation [2••,23,24]. DivJ is able to efficiently phosphorylate DivK but not CtrA in vitro [2••] and it contributes substantially to DivK phosphorylation in vivo
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[25••]. In addition, a mutant ctrA allele (sokA) suppresses the lethality phenotype of a divK null mutation [2••]. These data postulate the presence of a phosphorelay [20] with the phosphoryl group transferred from DivJ to the phosphotransfer protein DivK and from there via a still unknown component to CtrA (Figure 2). The sokA allele can also bypass the conditional cell division phenotype of cold-sensitive divL mutants [21••]. The DivL kinase is essential for cell viability and instead of being phosphorylated on a histidine residue, autophosphorylates on a tyrosine residue [21••]. DivL can catalyze CtrA phosphorylation and activation in vitro, suggesting that this kinase controls CtrA via a DivJ/DivK-independent pathway (Figure 2). A third signal transduction pathway leading to the activation of CtrA is defined by the CckA histidine kinase (Figure 2). CckA is essential for C. crescentus growth and is required for CtrA phosphorylation in vivo [22••]. CckA belongs to the group of hybrid kinases that contain both a kinase and a receiver domain of response regulators [26]. The CckA receiver domain could be involved in phosphotransfer from the kinase domain to an as yet unidentified phosphotransferase as the link between CckA and CtrA. Alternatively, the phosphoryl group could be transferred directly from CckA to CtrA with the CckA receiver domain playing a regulatory role in this process. It is interesting to note that out of 51 different histidine kinases encoded by the C. crescentus chromosome at least 20 are hybrid kinases. This compares with five hybrid kinases among 29 reported histidine kinases in E. coli [27]. It can be anticipated that the complexity of this regulatory network will increase and that additional signal transduction components such as phosphotransfer proteins or phospho-protein phosphatases will be found to regulate CtrA activity. But why are multiple non-redundant pathways required for the regulation of one common response regulator? It is likely that this regulatory network has to integrate a multitude of different structural and physiological signals before it ‘allows’ CtrA to make a number of very vital ‘decisions’ during cell propagation.
Differential localization of the CtrA sensor kinases The most fascinating aspect of the CtrA signal transduction pathway is provided by the recent observation that several of the sensor kinases undergo dynamic rearrangement during the cell cycle and localize to specific subcellular sites. Just as with most sensor protein kinases, CckA is membrane bound and evenly distributed throughout most of the cell cycle. However, in the early predivisional cell, coinciding with the onset of CtrA phosphorylation, CckA concentrates at the pole opposite the stalk [22••] (Figure 3). Polar positioning of CckA is maintained until immediately before the cells divide. Initial evidence suggests that polar localization is required for CckA activity and phosphorylation of CtrA [22••]. The histidine kinase PleC is distributed evenly in the membrane
of stalked and early predivisional cells before it dynamically rearranges to the pole that carries the flagellum and the chemosensory proteins [25••] (Figure 3). During the G1 to S transition, PleC delocalizes and the DivJ kinase concentrates at the stalked pole (Figure 3). PleC controls polar morphogenesis and motility [28], and is required for polar localization and possibly the activation of DivJ [25••]. The finding that these sensory components specifically localize to the Caulobacter cell poles at distinct stages of the cell cycle suggests that CtrA activity is controlled at least in part by the nature of the poles. Every newly formed pole at cell division develops into a flagellated swarmer pole and then into a stalked pole. The fact that poles are ‘marked’ through development by successive positioning of different kinases, together with the finding that positioning of a given kinase might be dependent on the activity or positioning of the preceding kinase, suggests that the different phosphorylation pathways, rather than acting on CtrA in parallel, are in fact interdependent pathways of an intricate regulatory network.
Concluding remarks: are checkpoints involved in cell cycle control? Although the mechanisms and components used to control CtrA levels and activity are currently being unraveled, the nature of the cell cycle signals that elicit the CtrA response remain unknown. As an attractive model one can postulate checkpoints that would time and coordinate different events as the cell cycle proceeds. But is there evidence for checkpoint control in Caulobacter? An ideal timing device would be the methylation status of the chromosome. Disrupting the normal cell cycle methylation pattern has indeed drastic consequences for cell growth and replication control [4,6]. In addition, there is evidence that CcrM-dependent DNA methylation contributes to the cell cycle transcription profile of the ccrM gene [5,29]. Thus, DNA methylation acts as a timing device in the Caulobacter cell cycle and additional events might exist that rely on this signal for correct temporal control. DNA replication could also act as cell cycle checkpoint. Inhibition of DNA replication in C. crescentus leads to a rapid loss of the promoter activity of early flagellar genes and the ccrM gene [5,30,31]. Because all of these promoters are controlled by CtrA, CtrA activity itself might be dependent on active DNA replication. Thus, CtrA could control replication, and in turn be controlled by replication in a feedback process. Recently, chromosome segregation has been added to the list of possible cell cycle checkpoints. The Caulobacter Cori regions are dynamically positioned to the cell poles immediately after the initiation of DNA replication [32•]. Proteins that are involved in this process include ParA, ParB and SMC [32•,33]. ParB binds to a region of the chromosome close to the origin and, together with ParA, was proposed to be involved in directing the movement of the DNA to the cell poles [33]. A null mutation in the smc gene is
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conditionally lethal and at the permissive conditions causes mislocalized origins and abnormal nucleoid morphology. When grown under restrictive conditions, the smc mutant arrests as predivisional cells with two fully replicated chromosomes [32•]. Interestingly, the blocked mutant cells fail to build up CtrA levels, suggesting that either CtrA synthesis is not turned on or CtrA degradation never turned off under these conditions [32•]. Thus, the correct structure and/or positioning of the chromosome could act as a checkpoint early in the S phase and in response trigger the increase of CtrA levels in the predivisional cell. One possibility is that the movement of proteins associated with the origin of replication to the poles constitutes a localized signal for later steps in the cell cycle. This brings us right back to the cell poles. Future work will have to be directed towards understanding the role of the poles as part of the regulatory network controlling Caulobacter cell cycle and development.
Acknowledgements The authors thank P Aldridge, C Stephens and RT Wheeler for critically reading the manuscript. We thank Y Brun for communicating results prior to publication. U Jenal is supported by the Swiss National Science Foundation grant 31-46764.96.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
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2. ••
Wu J, Ohta N, Newton A: An essential, multicomponent signal transduction pathway required for cell cycle regulation in Caulobacter. Proc Natl Acad Sci USA 1998, 95:1443-1448. The authors present genetic evidence that the essential single-domain response regulator DivK is involved in CtrA activation. A biochemical analysis demonstrates that the sensor kinase DivJ phosphorylates DivK preferentially over CtrA. The authors also show that the phosphorylated form of CtrA is required for in vitro transcription of a flagellar gene. This work postulates that a multicomponent phosphorelay is involved in CtrA activation in Caulobacter. 3. •
Reisenauer A, Quon K, Shapiro L: The CtrA response regulator mediates temporal control of gene expression during the Caulobacter cell cycle. J Bacteriol 1999, 181:2430-2439. The authors show that the differential temporal control of CtrA-regulated genes during the cell cycle can in part be explained by the different affinity of phosphorylated CtrA for their promoters, thus correlating the timing of transcription with the cellular levels of CtrA. 4.
Stephens C, Reisenauer A, Wright R, Shapiro L: A cell cycleregulated bacterial DNA methyltransferase is essential for viability. Proc Natl Acad Sci USA 1996, 93:1210-1214.
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6.
Wright R, Stephens C, Zweiger G, Shapiro L, Alley MR: Caulobacter Lon protease has a critical role in cell-cycle control of DNA methylation. Genes Dev 1996, 10:1532-1542.
7. •
Kelly AJ, Sackett MJ, Din N, Quardokus E, Brun YV: Cell cycle dependent transcriptional and proteolytic regulation of FtsZ in Caulobacter. Genes Dev 1998, 12:880-893. This work provides the regulatory basis for the observation that the cytokinesis protein FtsZ is not present in non-replicating swarmer cells. The cell cycle control of FtsZ includes CtrA-regulated expression and cell type-specific proteolysis. Newly synthesized FtsZ is stable when it is assembled at mid-cell at the beginning of the cell cycle. FtsZ degradation seems to be coupled to its disassembly after cell division.
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Sackett MJ, Kelly AJ, Brun YV: Ordered expression of ftsQA and ftsZ during the Caulobacter crescentus cell cycle. Mol Microbiol 1998, 28:421-434. This paper demonstrates that the ftsQA genes are transcribed independently of and in sequence with ftsZ during the cell cycle. 9.
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10. Quon KC, Yang B, Domian IJ, Shapiro L, Marczynski GT: Negative •• control of bacterial DNA replication by a cell cycle regulatory protein that binds at the chromosome origin. Proc Natl Acad Sci USA 1998, 95:120-125. The authors describe the mechanisms of DNA replication control by CtrA. CtrA represses the initiation of DNA replication by binding to five CtrA boxes inside the origin of replication. This restricts replication initiation to the phase of the cell cycle when CtrA is not present in the cell. 11. Marczynski GT, Lentine K, Shapiro L: A developmentally regulated chromosomal origin of replication uses essential transcription elements. Genes Dev 1995, 9:1543-1557. 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. Domian IJ, Reisenauer A, Shapiro L: Feedback control of a master • bacterial cell-cycle regulator. Proc Natl Acad Sci USA 1999, 96:6648-6653. This work explains the mechanisms of ctrA transcriptional autoregulation. Two ctrA promoters are active in the early and late predivisional cells, respectively. The first is repressed by CtrA and thus activated after the initiation of DNA replication when the regulator is absent. The accumulation of CtrA will then activate the second promoter resulting in high CtrA levels in late predivisional cells. 14. Jenal U, Fuchs T: An essential protease involved in bacterial cell • cycle control. EMBO J 1998, 17:5658-5669. The authors demonstrate that the Caulobacter ClpXP protease is required for cell cycle progression and for CtrA degradation during the G1 to S transition. 15. Østerås M, Stotz A, Schmid Nuoffer S, Jenal U: Identification and transcriptional control of the genes encoding the Caulobacter crescentus ClpXP protease. J Bacteriol 1999, 181:3039-3050. 16. Zhou Y, Gottesman S: Regulation of proteolysis of the stationaryphase sigma factor RpoS. J Bacteriol 1998, 180:1154-1158. 17.
Tatsuta T, Tomoyasu T, Bukau B, Kitagawa M, Mori H, Karata K, Ogura T: Heat shock regulation in the ftsH null mutant of Escherichia coli: dissection of stability and activity control mechanisms of sigma32 in vivo. Mol Microbiol 1998, 30:583-593.
18. Turgay K, Hahn J, Burghoorn J, Dubnau D: Competence in Bacillus subtilis is controlled by regulated proteolysis of a transcription factor. EMBO J 1998, 17:6730-6738. 19. Becker G, Klauck E, Hengge-Aronis R: Regulation of RpoS proteolysis in Escherichia coli: the response regulator RssB is a recognition factor that interacts with the turnover element in RpoS. Proc Natl Acad Sci USA 1999, 96:6439-6444. 20. Perraud AL, Weiss V, Gross R: Signalling pathways in twocomponent phosphorelay systems. Trends Microbiol 1999, 7:115-120. 21. Wu J, Ohta N, Zhao J, Newton A: A novel bacterial tyrosine kinase •• essential for cell division and differentiation. Proc Natl Acad Sci USA 1999, 96:13068-13073. This paper shows that the sensor kinase, DivL, which autophosphorylates on a tyrosine instead of the usual histidine residue, can phosphorylate CtrA in vitro. The divL gene is essential for viability of Caulobacter. The lethal phenotype of a divL mutant can be suppressed by a mutant allele of ctrA linking the DivL sensor to the CtrA regulatory network. 22. Jacobs C, Domian IJ, Maddock JR, Shapiro L: Cell cycle-dependent •• polar localization of an essential bacterial histidine kinase that controls DNA replication and cell division. Cell 1999, 97:111-120. This paper describes the identification of CckA, a sensor kinase required for phosphorylation of CtrA in vivo. CckA is a membrane-bound protein that localizes to the swarmer pole in the early predivisional cell, but is evenly distributed during the rest of the cell cycle. Evidence is presented suggesting that correct localization of CckA is important for its activity. 23. 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.
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24. Ohta N, Lane T, Ninfa EG, Sommer JM, Newton A: A histidine protein kinase homologue required for regulation of bacterial cell division and differentiation. Proc Natl Acad Sci USA 1992, 89:10297-10301. 25. Wheeler R, Shapiro L: Differential localization of two histidine •• kinases controlling bacterial cell differentiation. Mol Cell 1999, 4:683-694. The two sensor kinases, PleC and DivJ, show a distinct pattern of subcellular localization during the cell cycle. PleC is localized to the swarmer pole of late predivisional cells and is replaced at this site by DivJ during the swarmer to stalk transition. Interestingly, correct localization of DivJ requires PleC. 26. Appleby JL, Parkinson JS, Bourret RB: Signal transduction via the multi-step phosphorelay: not necessarily a road less traveled. Cell 1996, 86:845-848. 27.
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29. Reisenauer A, Kahng LS, McCollum S, Shapiro L: Bacterial DNA methylation: a cell cycle regulator? J Bacteriol 1999, 181:51355139. 30. Dingwall A, Zhuang WY, Quon K, Shapiro L: Expression of an early gene in the flagellar regulatory hierarchy is sensitive to an interruption in DNA replication. J Bacteriol 1992, 174:1760-1768. 31. Stephens CM, Shapiro L: An unusual promoter controls cell-cycle regulation and dependence on DNA replication of the Caulobacter fliLM early flagellar operon. Mol Microbiol 1993, 9:1169-1179. 32. Jensen RB, Shapiro L: The Caulobacter crescentus smc gene is • required for cell cycle progression and chromosome segregation. Proc Natl Acad Sci USA 1999, 96:10661-10666. The authors used fluorescence in situ hybridization to show that the Caulobacter origins of replication move to opposite cell poles immediately after initiation of replication. A conditionally lethal smc null mutant results in abnormal DNA positioning and cell cycle arrest at the predivisional stage. Arrested smc mutants complete DNA replication but fail to build up CtrA levels. This observation proposes a segregation checkpoint upstream of CtrA activity in the predivisional cell. 33. Mohl DA, Gober JW: Cell cycle-dependent polar localization of chromosome partitioning proteins in Caulobacter crescentus. Cell 1997, 88:675-684.