Abstract
The CtrA master transcriptional regulator is a central control element in Caulobacter cell cycle progression and polar morphogenesis. Because of its critical role, CtrA activity is temporally regulated by multiple mechanisms including phosphorylation and ClpXPdependent degradation of CtrA. The CckA histidine kinase is known to contribute to CtrA phosphorylation. We show here that genes differentially expressed in a ctrA temperature-sensitive (ts) mutant are similarly affected in a cckA ts mutant, that the phosphorylation of CckA coincides temporally with CtrA phosphorylation during the cell cycle, and that CckA is essential for viability because it is required for CtrA phosphorylation. Thus, it is the signal transduction pathway mediated by CckA that culminates in CtrA activation, which is temporally regulated and essential for cell cycle progression. CckA also positively regulates CtrA activity by a mechanism that is independent of CtrA phosphorylation. CtrA is more stable in the presence of CckA than it is absence, suggesting that CckA may also be involved, directly or indirectly, in the regulation of CtrA proteolysis.
Introduction
Two-component signal transduction proteins play a critical role in the control of cell cycle progression and polar morphogenesis in the bacterium Caulobacter crescentus . A small number of these regulatory proteins control the expression of a large portion of cell cycle-regulated genes. The CtrA master transcriptional regulator controls multiple cell cycle events and couples polar morphogenesis with cell cycle progression (Quon et al ., 1996; Kelly et al ., 1998; Quon et al ., 1998; Laub et al ., 2000; Skerker and Shapiro, 2000; Laub et al ., 2002) . In addition, CtrA controls the initiation of chromosome replication by directly binding to the origin of replication, preventing replisome formation in G1 cells (Quon et al ., 1998) .
Introduction
Tight control of CtrA activity is critical to the coordinated progression of cell cycle events and to cellular morphogenesis ( Fig. 1) . In the swarmer cell, the chromosome replication origin is silenced by the binding of the phosphorylated form of CtrA (CtrA ~ P), but as the swarmer cell differentiates into a stalked cell, CtrA ~ P is degraded by the ClpXP protease complex (Jenal and Fuchs, 1998) , allowing initiation of chromosome replication (Domian et al ., 1997; Quon et al ., 1998) . After the initiation of replication in the stalked cell, CtrA is synthesized de novo and is immediately phosphorylated, presumably to prevent further rounds of replication initiation (Quon et al ., 1996; Domian et al ., 1997) . In the predivisional cell, CtrA ~ P activates the cascade of flagellar gene transcription and the expression of the ccrM gene encoding an essential DNA methyltransferase (Quon et al ., 1996) . The CtrA protein is preferentially cleared from the stalked compartment of the late predivisional cell, allowing the initiation of replication in the progeny stalked cell, while maintaining CtrA in the progeny swarmer cell where it functions to prevent the initiation of DNA replication (Domian et al ., 1997) .
Introduction
Whereas much is known about when CtrA is present and active during the cell cycle, very little is understood about how its regulation is accomplished. As expected for the response regulator family of proteins, the activity of CtrA is controlled by phosphorylation on the conserved aspartate residue (Asp51) (Quon et al ., 1996) . Phosphorylation of CtrA is essential for cell viability and greatly enhances its DNA binding and the consequent transcriptional activity of its target genes in vitro (Quon et al ., 1998; Wu et al ., 1998; Domian et al ., 1999) . Three signal transduction pathways have been proposed to control CtrA phosphorylation levels (Wu et al ., 1998; Jacobs et al ., 1999; Wu et al ., 1999; Osteras and Jenal, 2000) . One of the putative pathways involves the histidine kinase, CckA (Jacobs et al ., 1999) . We showed previously that a cckA temperature-sensitive (ts) mutant ( cckATS1 ) is essential for viability and exhibits a pleiotropic phenotype, including defects in flagella and pili biogenesis, and initiation of DNA replication and cell division, that are characteristic of a ctrA loss-of-function mutant (Jacobs et al ., 1999) . CtrA phosphorylation was shown to be dramatically reduced in cckATS1 cells. The cckA gene encodes a hybrid histidine kinase that contains a receiver domain at its C-terminus with the conserved aspartate phosphorylation site usually found in response regulators. This hybrid structure is not uncommon (Grebe and Stock, 1999) , and suggests that CckA may be part of a multicomponent phosphorelay.
Introduction
We report here that CckA is essential for CtrA phosphorylation during the cell cycle and that, in the absence of CckA, CtrA is inactive. Phosphorylation of both CckA and CtrA is temporally controlled and coincident, reflecting the pivotal role of CckA-mediated signal transduction in Caulobacter cell cycle progression. CckA has an additional function that is independent of CtrA phosphorylation. We present data suggesting that this additional function may be the protection of CtrA from proteolysis.
Results

The CckA histidine kinase and the CtrA response regulator control the same genetic pathways
The essential CckA histidine kinase has been proposed to play a role in the phosphorylation of CtrA. The most compelling evidence is that CtrA phosphorylation is dramatically reduced in the cckATS1 mutant (Jacobs et al ., 1999) . If CckA function mainly resides in the regulation of CtrA activity, the expectation is that CckA controls CtrAdependent genes or a subset of them. Therefore, we used whole-genome microarrays and two-dimensional gel electrophoresis to compare the respective RNA and protein levels of ts strains mutant for CtrA or CckA function. Three different strains were used: wild-type CB15N, ctrAV148F and cckATS1. The ctrA and cckA ts mutant strains were isolated in the same genetic screen (Jacobs et al ., 1999) . All three strains were grown in rich media at 28 ∞ C (permissive temperature for viability) to early log phase and then shifted to 37 ∞ C (restrictive temperature) for 4 h. The cell morphology and DNA content of each strain after 4 h at 37 ∞ C are shown in Fig. 2A and B. Strains bearing ts alleles of ctrA and cckA exhibit similar phenotypes after a 4 h shift to 37 ∞ C (Jacobs et al ., 1999) . They show excessive filamentation, accumulation of multiple chromosomes per cell, and an eventual loss in viability with follows similar kinetics (data not shown). RNA from each of the ts strain cultures, grown at 28 ∞ C in rich media and then shifted to 37 ∞ C for 0, 2 and 4 h, was obtained and compared to RNA from the wild-type strain, grown under the same conditions, using whole-genome microarrays. The entire experiment was repeated twice and results were averaged (see Supplementary material , 'Microarray ctrA cckA ts series'). We then selected the cell cycle-regulated genes (Laub et al ., 2000) whose expression level changed greater than twofold in either of mutant strains. The expression profiles for 212 genes at the 4 h time-point is shown in Fig. 2C . They are nearly identical for ctrAV148F and cckATS1 , with a correlation coefficient of 0.91 for the genes shown. This level of correlation is on par with that seen when comparing two RNA samples from the same strain, and so the differences between the ctrAV148F and cckATS1 profiles is probably due to experimental noise. As an additional test of profile similarity, we averaged data from the first replicate of ctrAV148F and the second replicate of cckATS1 and compared that to an average of the second replicate of ctrAV148F and the first replicate of cckATS1 . The correlation coefficient for such a comparison is 0.8, indicating that the profiles amongst all replicates, and hence, between each strain, are very similar. These results support the notion that CckA and CtrA are in the same signalling pathway. Finally, for the genes that were previously identified as direct targets of CtrA (Laub et al ., 2002) , we note here that all of these genes were found to be significantly changed in the ctrAV148F strain and ~ 95% were also found to be strongly affected in the cckATS1 strain (data not shown).
The CckA histidine kinase and the CtrA response regulator control the same genetic pathways
To determine whether loss of CckA function also causes CtrA-dependent effects at the translational or post-translational levels, the global protein profiles of ctrAV148F and cckATS1 cell cultures after 4 h at 37 ∞ C were examined in comparison to wild-type CB15N cells grown under the same conditions (see Supplementary material , Fig. S1 ). Total cell extracts were separated using two-dimensional gel electrophoresis and the resulting images were compared to detect proteins with different levels. In total, 30 differentially abundant proteins, defined by a minimum twofold change in spot abundance and replicated across three independent gel series, were detected (see Supple- mentary material , Fig. S1 for gels, and Table S1 for protein spot identity). Out of this set, only two spots (identified as CC2258, heat-shock protein Hsp20 family and CC3494, OmpA family protein) appeared to be down-or upregulated in one of the mutants but not in the other (see Supplementary material , Table S1 ). Thus, the protein level profiles, like the gene transcript profiles, are very similar between ctrA and cckA mutants supporting the argument that CckA function is mediated through CtrA.
CckA phosphorylation during the cell cycle correlates with that of CtrA
CtrA is phosphorylated in swarmer and predivisional cells, but not in stalked cells (Domian et al ., 1997) . This was demonstrated using a proteolysis-resistant form of CtrA. As the phosphorylated form of CtrA (CtrA ~ P) has a short half-life (under 5 min) relative to the length of the cell cycle (Domian et al ., 1997) , an active signal transduction pathway must continuously phosphorylate CtrA in swarmer and predivisional cells, but not in stalked cells. If CckA contributes to the regulation of CtrA phosphorylation throughout the cell cycle, one would expect that the times of its activity would correlate with the times when CtrA phosphate is present. Alternatively, CckA may be involved in CtrA phosphorylation only at specific times during the cell cycle, but hcarey@example.com. Thus, to determine when CckA acts upon CtrA phosphorylation during the cell cycle, we sought to determine when CckA is active. Histidine kinases form dimers that act as autokinases with each subunit catalysing the transfer of a phosphoryl group from ATP to the conserved histidine of the other subunit upon signal activation (Grebe and Stock, 1999; Hoch, 2000) . The phosphoryl group is subsequently transferred onto the conserved aspartate phosphorylation site of the cognate response regulator. Thus, the phosphorylation state of histidine kinases is usually considered a valid measure of their activity.
CckA phosphorylation during the cell cycle correlates with that of CtrA
To determine when the CckA histidine kinase is phosphorylated during the cell cycle and how its phosphorylation pattern correlates with that of CtrA phosphorylation, we performed in vivo phosphorylation experiments on synchronized populations of wild-type CB15N cells. In this assay, both CtrA and CckA were immunoprecipitated after radiolabelling with 32 Pi to assess their phosphorylation C. Expression profiles for ctrAV148F , and cckATS1 4 h after a shift to restrictive temperature (37 ∞ C). Expression data is shown for 212 cell cycleregulated genes (Laub et al ., 2000) whose expression level changes greater than twofold in either of the mutant strains. Changes in mRNA levels relative to a common reference derived from wild-type cells are represented using the colour scale shown at the bottom. levels at specific times during the cell cycle (Fig. 3A) . CckA has two predicted phosphorylation sites (His322 in the transmitter domain and the Asp623 in the C-terminal receiver domain) that cannot be distinguished by this technique. We performed the in vivo phosphorylation assay three times and, in each case, the phosphorylation level of the CckA histidine kinase was found to be highest in predivisional cells (around 110 min) and lowest in stalked cells (around 50 min) (Fig. 3B) , following a temporal pattern parallel to that of CtrA phosphorylation (Fig. 3C ). The coincidence of CckA and CtrA phosphorylation levels during the cell cycle suggests that the CckA histidine kinase controls CtrA phosphorylation throughout the cell cycle.
Besides its essential role in CtrA phosphorylation, CckA has another function that is important for normal cell cycle progression
Mutation of the phosphorylation site of CtrA from an aspartate residue to a glutamate residue (CtrAD51E) has been shown to result in constitutive, although partial, phosphorylation-independent activity (Domian et al., 1997) . This ctrAD51E mutant allele, when expressed in high copy number (pCtrAD51E), supports viability in a DctrA strain in the absence of phosphorylation (Domian et al., 1997) . We reasoned that if the CckA histidine kinase was essential for viability because of its requirement for CtrA phosphorylation, the cckA gene may become dispensable in the presence of the functional phosphorylation-independent CtrAD51E. Using a transducing phage lysate carrying C. crescentus chromosomal sequences in which most of the cckA sequence had been replaced by the gentamycin resistance gene cassette (Dcck888a:dbb7:2d49:b3f6:8605:bf4e:caed:e5a4Gent R ), we confirmed our previous observation (Jacobs et al., 1999) that chromosomal disruptions of cckA do not occur in wild-type CB15N unless a second functional copy of cckA is present (Table 1) . We used the same lysate to transduce wild-type CB15N or DctrA strains expressing either wild-type ctrA or ctrAD51E from a high copy number plasmid (pCtrA and pCtrAD51E respectively). Consistent with our hypothesis, Gent R transductants were only recovered in strains overexpressing the phosphorylationindependent CtrAD51E mutant creating CJ395 (DcckA/ pCtrAD51E) and CJ396 (DctrADcckA/pCtrAD51E) but none were recovered in strains expressing wild-type ctrA from the same high copy number plasmid (Table 1) . Immunoblot blot analysis confirmed the chromosomal disruption of cckA in CJ396 and CJ395 as no CckA protein was detected (Fig. 4A , lanes 4 and 5). Strains like wild-type CB15N, CB15N/pCtrAD51E, and LS2716 (DctrA/ pCtrAD51E), which all carry cckA on the chromosome, were used as controls (Fig. 4A) .
Besides its essential role in CtrA phosphorylation, CckA has another function that is important for normal cell cycle progression
In vivo phosphorylation experiments with the same strains showed that CtrAD51E was not phosphorylated in either DctrA or DcckA strains (Fig. 4B, lanes 3 and 4) . This is consistent with previous in vivo and in vitro studies showing that CtrAD51E mutant protein cannot be phosphorylated (Domian et al., 1997; Reisenauer et al., 1999) . The phosphorylation level of wild-type CtrA was reduced in CB15N/pCtrAD51E relative to wild-type CB15N (Fig. 4B, lane 2 and 1 respectively) . This effect is probably due to the known autoregulation of ctrA expression . In CB15N/pCtrAD51E, the multicopy number of pCtrAD51E results in overexpression of ctrAD51E, which probably inhibits to some extent the synthesis of wild-type CtrA from the chromosome, and therefore the level of CtrA~3 2 P. This presumably also occurs for the CJ395 strain (DcckA/pCtrAD51E). It was, however, interesting that no CtrA~3 2 P could be detected in the latter strain even though it still carries an intact copy of wild-type ctrA on the chromosome (Fig. 4B , lane 5 to be compared with lane 2). To ascertain that CtrA protein was indeed present in this background, the chromosomal ctrA gene was replaced by a tagged copy of ctrA, ctrAD3M2 whose product is known to be under a normal phosphorylation control when CckA is present but, unlike wild-type CtrA, can be distinguish electrophoretically from CtrAD51E because of its size difference . In this DcckA strain (ctrAD3M2;DcckA/pCtrAD51E), CtrAD3M2 protein was clearly present as shown by immunoblot ( Fig. 4C; lane 6 ), yet no phosphorylation could be detected in an in vivo phosphorylation assay (Fig. 4D , lane 6). This observation is of interest since other histidine kinases besides CckA have been proposed to contribute to CtrA phosphorylation (Wu et al., 1998; . One might have expected that in the absence of CckA, the activity of these other histidine kinases would have contributed to some residual phosphorylation of CtrA. It is possible that the phosphorylation of CtrA by CckA-independent pathways resides below the detection level of our technique. Alternatively, the other proposed CtrA phosphorylation pathways may be dependent on CckA function. In any case, the fact that the phosphorylationindependent CtrAD51E mutant protein can support viability in the absence of CckA when the wild-type CtrA cannot (Table 1 ) provides strong genetic evidence that the CckA histidine kinase is essential for viability because it is required for CtrA phosphorylation.
Besides its essential role in CtrA phosphorylation, CckA has another function that is important for normal cell cycle progression
Interestingly, the DcckA strains (CJ395 and CJ396) exhibited parallel phenotypes, including severe filamentation, accumulation of chromosomes (Fig. 5A ) and lysis at high cell density. These phenotypes were absent in LS2716 cells (DctrA/pCtrAD51E). As CtrAD51E does not require phosphorylation for its activity (Domian et al., 1997) , we reasoned that if the sole function of CckA was to regulate CtrA phosphorylation, LS2716 and CJ396 (DctrA/pCtrAD51E and DctrADcckA/pCtrAD51E respectively) should have the same phenotypes. Their phenotypic differences suggest that in addition to mediating the phosphorylation of CtrA, the CckA histidine kinase has another function that is critical for cell cycle progression.
The additional role of CckA appears to be a phosphorylation-independent activation of CtrA
To directly examine if the additional CckA function that is independent of CtrA phosphorylation affects gene expression, we compared the RNA levels of cell cycle-regulated genes between LS2716 (DctrA/pCtrAD51E) and CJ396 (DctrADcckA/pCtrAD51E). Total RNA from each strain was analysed on whole-genome microarrays and compared with RNA from wild-type CB15N cells grown under the same conditions. These comparisons were done in triplicate and results averaged (see Supplementary material, 'Microarray CtrAD51E series'). Out of the 3536 genes for which reliable data were available, 226 (6%) were induced or repressed greater than twofold in CJ396 relative to LS2716. Many of the observed expression changes may be a reflection of the organism compensating for its perturbed cell cycle. To filter out these expression differences and focus on the additional role CckA plays in controlling cell cycle progression, only the genes that are known to be cell cycle-regulated (Laub et al., 2000) were selected for further examination. The relative RNA levels for each of 544 cell cycle-regulated genes were compared for the B. The RNA levels of LS2716 (DctrA/pCtrAD51E) and CJ396 (DctrADcckA/pCtrAD51E) were compared, on whole-genome microarrays, to a common reference RNA derived from wild-type cells (see Experimental procedures). The ratios for all cell cycle-regulated genes (544 in total) were then plotted against each other as a scatterplot. Genes falling above or below the parallel lines had expression levels at least twofold higher in one strain compared with the other. C. Comparison of RNA levels, performed as in (B), between CJ396 (DctrADcckA/pCtrAD51E) and CJ395 (DcckA/pCtrAD51E).
The additional role of CckA appears to be a phosphorylation-independent activation of CtrA
two strains (shown as scatterplots in Fig. 5B) . Consistent with the dramatic phenotypic differences between the two strains ( Fig. 5A) , the gene expression profiles were also significantly different. Out of these, 47 genes were repressed and 10 genes were induced in CJ396 relative to LS2716 (a list of them is provided as Supplementary material ). These two strains are isogenic except for the presence or absence of a functional copy of cckA. We anticipated that the functions and identities of these genes would help unravel the additional role CckA plays in governing cell cycle progression. Surprisingly, inspection of this list revealed that of these 57 genes whose expression was significantly altered between the two strains, 55 of them (96%) were similarly induced or repressed in the ctrA ts mutants relative to wild-type (see Supplementary material, Gene list). The fact that nearly all of these genes are CtrA-dependent suggests that CckA's additional role is a phosphorylation-independent activation of CtrA. However, we cannot rule out the possibility that CckA participates in an additional signal transduction pathway that regulates, independently of CtrA, this particular subset of CtrA-dependent genes.
In the absence of CckA, CtrA is inactive
The fact that the presence of wild-type ctrA in CJ395 (DcckA/pCtrAD51E) strain does not rescue or suppress the severity of the cell division and DNA replication phenotypes (Table 1 ; Fig. 5A ) seen in CJ396 (DctrADcckA/ pCtrAD51E) suggests that in the absence of CckA, CtrA is inactive. To obtain a sensitive readout of any putative CtrA activity in CJ395, we performed a comparative gene expression analysis between CJ395 and CJ396 (which only differ by the presence or absence of a functional copy of ctrA on the chromosome). Each strain was grown to mid-log phase and total RNA was harvested for comparison, on whole-genome microarrays, to RNA from wildtype CB15N cells grown under the same conditions. These comparisons were carried out in triplicate and the results were averaged (see Supplementary material, 'Microarray CtrAD51E series'). As with other comparisons, we analysed only those genes shown previously to be cell cycle regulated. The relative RNA levels for each cell cycle-regulated gene on the microarrays were then compared for the two strains (shown as a scatterplot, Fig. 5C ), resulting in a correlation coefficient of 0.97. Only three out of 544 cell cycle-regulated genes (CC0782 transcriptional regulator of LuxR family, CC0707 conserved hypothetical protein and CC0559 hypothetical protein) showed greater than twofold induction or repression, and they probably represent noise or false positives. Thus, in agreement with the observed cell cycle defects, the gene expression profiles of the DcckA strains, CJ395 and CJ396, are nearly indistinguishable despite the fact that one strain carries an intact copy of ctrA on the chromosome. These data, together with the fact that no CtrA phosphorylation can be detected in cckA deletion strains (Fig. 4B , lane 5 relative to lane 2 and Fig. 4D , lane 6 relative to lane 2), suggest that in the absence of CckA, CtrA is unphosphorylated and inactive.
CckA appears to be also involved in the control of CtrA stability
The DNA microarray analysis of CJ396 transcripts relative to LS2716 (see above) suggest that CckA's additional role in cell cycle control is a positive regulation of CtrA. If correct, what could be the molecular basis for this phosphorylation-independent regulation of a response regulator by a histidine kinase? An important aspect of CtrA regulation is its cell cycle-regulated proteolysis (Domian et al., 1997) . One possibility is that CckA may affect the stability of CtrA and thereby alter CtrA activity, regardless of the phosphorylation state. This possibility was suggested by the immunoblots shown in Fig. 4A . CtrAD51E protein levels were lower in DcckA strains (lanes 4 and 5 relative to lanes 3 and 2 respectively). As only high levels of CtrAD51E proteins, but not low levels, can support viability in a DctrA strain (Domian et al., 1997) , a decrease in CtrAD51E protein levels could be sufficient to explain the phenotypic defects observed in DcckA strains. In order to test if CckA affects CtrAD51E stability, we determined the half-life of CtrAD51E and the proteolysis resistant mutant protein CtrAD3M2 in two genetic backgrounds that only differ by the presence or absence of CckA. The first strain is CJ507 in which the wild-type ctrA is replaced by ctrAD3M2 (an allele whose product is resistant to proteolysis at the G1-S transition) on the chromosome (ctrAD3M2/pCtrAD51E). The second strain, CJ508, is genetically identical except for the chromosomal deletion of the cckA gene (ctrAD3M2;DcckA/pCtrAD51E). Both strains harboured the multicopy pCtrAD51E plasmid. The CJ507 strain had wild-type cell morphology whereas the CJ508 strain had the characteristic filamentation phenotype of DcckA strains (data not shown). Because of the cell cycle defects seen in DcckA strains, we were concerned that an increased instability of CtrA in such strains may be due to induction of overall protein degradation. If true, we speculated that the degradation would not be specific and would similarly affect the G1-S proteolysis resistant CtrAD3M2 protein. Cultures of both strains were pulse-labelled with 35 S-methionine in early log phase, and samples were withdrawn at several times during the following 60 min. The CtrA derivatives were immunoprecipitated using anti-CtrA antibodies. As shown in Fig. 6A , the proteolysis resistant CtrAD3M2 was equally stable in the presence or absence of CckA, arguing that there is little or no induction of general protein degradation in the absence of CckA.
CckA appears to be also involved in the control of CtrA stability
We observed that the 35 S-labelled CtrAD51E levels decreased at a faster rate in the DcckA deletion strain (Fig. 6A) . As CtrAD3M2 stability was not significantly affected by the presence or absence of CckA, the 35 Slabelled CtrAD51E levels were analysed over time as an expression of the ratio of CtrAD51E and CtrAD3M2 levels to reduce handling and loading-induced errors. As shown in Fig. 6B , the relative CtrAD51E degradation rate was significantly higher in the DcckA strain (CJ508) when compared with the CJ507 strain, with half of the CtrAD51E protein level being degraded in approximately 15 min when CckA was absent and in 35 min when CckA was present. As ctrAD51E is only a partial functional allele of ctrA, which has to be overexpressed to support viability (Domian et al., 1997) , a higher turnover of its gene product in the absence of CckA provides an explanation for the phenotypes seen in the DcckA strains. It has been demonstrated that CtrAD51E is normally degraded during the G1-S transition, even when overexpressed from a high copy number plasmid (Domian et al., 1997) , suggesting that CckA histidine kinase may contribute, directly or indirectly, to the stability of the master response regulator CtrA during the cell cycle.
Discussion
Because of its central role, CtrA activity is tightly regulated by several mechanisms including temporally controlled synthesis, ClpXP-dependent degradation, and phosphorylation of CtrA (Quon et al., 1996; Domian et al., 1997; Jenal and Fuchs, 1998) . We present evidence here that the CckA histidine kinase plays a central role in the regulation of CtrA activity. This conclusion is based on several observations: (i) CtrA phosphorylation is dramatically reduced in a cckA loss-of-function mutant, suggesting that CckA controls CtrA phosphorylation (Jacobs et al., 1999) ; (ii) an analysis of the RNA and protein levels in ctrA and cckA ts mutants relative to wild-type shows that most genes that are differentially expressed in a ctrA ts mutant were similarly affected in the cckA ts mutant. The strong correlation in global gene expression profiles and protein levels between ctrA and cckA ts mutants indicates that a loss of CckA function corresponds in a loss of CtrA activity; (iii) the fact that CtrA phosphorylation itself, and not only the mere presence of the protein, is cell cycle regulated (Domian et al., 1997) predicts that the signal transduction pathway that activates CtrA follows the same cell cycle pattern as that of CtrA phosphorylation. In support of this, phosphorylation of CckA, and therefore presumably its activity, coincides temporally with CtrA phosphorylation during the course of the cell cycle. It suggests that CckA is involved in the regulation of CtrA phosphorylation at each stage of the cell cycle; (iv) only the phosphorylation-independent CtrAD51E mutant protein supports viability in the absence of CckA protein, providing strong genetic evidence that CckA is essential for viability because it is required for CtrA phosphorylation; and (v) finally, it appears that CckA has an additional role in controlling CtrA activity, that of stabilizing the CtrA protein.
Discussion
The expression of phosphorylation-independent CtrAD51E mutant protein from a multicopy plasmid allows the cells to live without a functional copy of cckA. However, in the absence of CckA, the cells have several phenotypes including severe filamentation and accumulation of chromosomes. The CtrAD51E protein cannot be phosphorylated in vitro or in vivo (Domian et al., 1997; Reisenauer et al., 1999) , and comparison of gene expression profiles using two DctrA strains with CtrAD51E that only differ by the presence or absence of CckA showed that most genes that were differentially expressed are known to be CtrA dependent. These results argue that CckA may affect CtrA activity by a means other than phosphorylation. In the absence of CckA, the stability of the CtrAD51E protein, which, like wild-type CtrA, is normally degraded during the swarmer-to stalked (G1-S) cell transition (Domian et al., 1997) , is significantly reduced. A reduced level in CtrAD51E proteins can easily explain the phenotypes observed in DcckA strains as elevated levels of CtrAD51E protein are necessary for its functionality (Domian et al., 1997) . When CckA interacts with its CtrA substrate, it may protect it from proteolysis by ClpXP. In Bacillus subtilis, it has been shown that the anti-sigma factor SpoIIAB not only phosphorylates the anti-anti sigma factor, SpoIIAA, but also form a complex with it that protects SpoIIAB from ClpCP-mediated proteolysis (Pan et al., 2001) . However, it is not known if CckA directly interacts with CtrA. Given its hybrid histidine kinase structure with a response regulator-like domain at its C-terminus, it is likely that CckA is involved in a phosphorelay with an unidentified histidine phosphotransferase as an intermediate between CckA and CtrA. Another possibility is that CckA may affect the phosphorylation state of another response regulator that directly controls the proteolysis of CtrA by ClpXP. There is precedent for regulated proteolysis by a signal transduction pathway. The RssB response regulator (also termed SprE or MviA) directly targets s S , the master regulator of the general stress response, to the ClpXP protease (Bearson et al., 1996; Muffler et al., 1996; Pratt and Silhavy, 1996) . RssB promotes s S degradation while having no effect on ClpXP activity with other substrates (Zhou and Gottesman, 1998) . RssB activity is modulated by phosphorylation suggesting that a histidine kinase, yet-tobe identified, controls s S proteolysis (Zhou et al., 2001 ). Finally, CckA may negatively regulate ClpXP protease activity irrespective of CtrA. However, we think that this possibility is unlikely because both clpX and clpP RNA levels were found to be twofold reduced in DcckA strains relative to wild-type CB15N (see Supplementary material 'Microarray CtrAD51E series') although we cannot rule out a post-transcriptional effect of CckA on the global ClpXP protease activity.
Discussion
Thus, we have presented evidence that CckA plays a critical role in the signal transduction pathway culminating in CtrA phosphorylation and it, at least partly, contributes to CtrA stability.
Discussion
Besides CckA, two other signal transduction pathways have been proposed to control CtrA phosphorylation levels (Wu et al., 1998; . Genetic and biochemical evidence clearly showed that PleC and DivJ histidine kinases control the activity of the essential single domain response regulator, DivK (Hecht et al., 1995; Burton et al., 1997; Wu et al., 1998; Wheeler and Shapiro, 1999) . A mutant ctrA allele (sokA) suppresses the lethality phenotype of a divK null mutant and the cell division defect of a divJ point mutant, providing a genetic link between the DivK signal transduction pathway and CtrA (Wu et al., 1998) . The sokA allele can also suppress the cell division phenotype of cold-sensitive point mutations in a gene encoding the essential DivL kinase suggesting that DivL also contributes to CtrA phosphorylation (Wu et al., 1999) . If several signal transduction pathways independently participate in increasing CtrA~P levels, one would expect that in the absence of CckA, the other pathways would contribute to a residual activation of CtrA. To address this question, we compared two DcckA strains, both containing CtrAD51E but differing by the presence or absence of wild-type ctrA on the chromosome. The two strains were undistinguishable; they were filamentous and they had multiple chromosomes and an identical altered profile of global gene expression. Thus, the presence of ctrA does not rescue any of the phenotypes even though the other putative CtrA signal transduction pathways are present. These results, together with the observation that no CtrA~P was detected in the DcckA strain with ctrA on the chromosome, argue that, in the absence of CckA, CtrA is unphosphorylated and inactive. To affect CtrA~P levels, the DivK and DivL signalling pathways may be genetically upstream of CckA or dependent on CckA function to be active. Another possibility is that these other signalling pathways negatively control CtrA activity by, for example, contributing to its dephosphorylation.
Discussion
Considering the pivotal role of CckA in differentiation and cell cycle control, the next critical question is: what mechanisms control CckA activity? The level of the CckA protein is constant during the cell cycle, suggesting that mechanisms using controlled proteolysis and/or synthesis are unlikely (Jacobs et al., 1999) . On the other hand, CckA phosphorylation is cell cycle controlled (Fig. 3) . Identifying the mechanisms regulating CckA phosphorylation, and therefore its activity, will be key. The striking observation that CckA changes its cellular location during the cell cycle suggests that this cell cycle-controlled localization may play an important role in CckA regulation (Jacobs et al., 1999) . We have shown that CckA is found around the entire cell membrane in swarmer and stalked cells. It then moves from this dispersed distribution to the cell pole in early predivisional cells, and disperses at cell division. The polar localization of CckA in predivisional cells coincides with the time of CckA peak phosphorylation. The integration of multiple temporal and spatial levels of CtrA activation mediated by the CckA histidine kinase yields a finely tuned regulatory pathway. The challenge is now to understand how a unicellular organism that lives in a dilute aquatic environment sends cues to signal transduction proteins to effect multiple cell cycle changes.
Experimental procedures

Bacterial strains, plasmids and growth
Strains and plasmids are listed in Table 2 . Caulobacter crescentus strains were routinely grown in peptone-yeast extract (PYE, complex media), M2G (minimal media) or M5GG (lowphosphate media) at 28∞C unless otherwise stated (Ely, 1991; Jacobs et al., 2001) . Plasmids were mobilized from Escherichia coli strain S17-1 into C. crescentus by bacterial conjugation (Ely, 1991) . Chromosomal disruptions of cckA were generated by FCR30 phage transduction (Ely, 1991) of Dcck888a:dbb7:2d49:b3f6:8605:bf4e:caed:e5a4Gent R into different C. crescentus strains carrying the ctrAD51E allele on a multicopy plasmid, pCtrAD51E (Jacobs et al., 1999) .
Flow cytometry
Samples for flow cytometry were prepared as described previously (Winzeler and Shapiro, 1995) and analysed in a Becton Dickinson FACStar Plus machine. Data were collected and analysed using FACS&SOL;DESK software (Stanford University, Stanford, CA).
Synchronization, immunoblots and immunoprecipitations
Homogenous populations of swarmer cells were isolated as described (Quon et al., 1996) , resuspended to A 660 of approximately 0.15, and allowed to proceed synchronously throughout the cell cycle. Samples were taken at different time intervals. Immunoblot and immunoprecipitations were performed as described (Jacobs et al., 1999) . CtrA stability was determined by performing a 5 min pulse with [
Synchronization, immunoblots and immunoprecipitations
35 S]-methionine and a chase with unlabelled methionine and casamino acid. At different time intervals after pulse, aliquots were lysed and immunoprecipitated with anti-CtrA serum as described previously (Domian et al., 1997 
In vivo phosphorylation
In vivo experiments were performed as described (Jacobs et al., 2001 ) with the following modifications: each sample was immunoprecipitated with 2 ml of anti-CtrA and 0.5 ml of anti-CckA antibodies.
Microscopy and photography
Cells were immobilized using a thin layer of agarose as described (Webb et al., 1998) . Normaski differential interference contrast (DIC) images were taken with a Princeton Micromax 5600 camera or a Hamamatsu ORCA-ER and processed by Metamorph (Universal Imaging).
DNA microarray, two-dimensional gel electrophoresis and data analysis
Peptone-yeast extract cultures were grown at 28∞C until they reached an A 660 of approximately 0.3. For the temperaturesensitive series of microarray experiments, PYE cultures of CB15N, ctrAV148F and cckATS1 were grown at 28∞C until they reached an A 660 of 0.1 (t = 0 h). The cultures were then shifted for 2 h at 37∞C (t = 2 h); at which time their A 660 was approximately 0.35. The cultures were then diluted four times with prewarmed PYE media and incubated at 37∞C for an Domian et al. (1997) pCtrAD51E pJS14 carrying ctrAD51E allele, also named pCTD14 Domian et al. (1997) additional 2 h (t = 4 h). RNA extraction was harvested for each sample and prepared for hybridization as described previously (Laub et al., 2002) . For the proteomic studies, 10 mg of freeze-dried C. crescentus cells were suspended in 1 ml two-dimensional buffer (5 M urea, 2 M thiourea, 0.1% carrier ampholytes pH 3-10 (Bio-Rad), 2% (w/v) CHAPS, 2% (w/v) sulphobetaine 3-10, 2 mM tributyl phosphine) and subjected to three rounds of tip-probe sonication for 30 s with 2 min on ice between each round. The lysate was centrifuged at 6000 g for 12 min to remove insoluble material. Then, 250 mg protein was used to perform each two-dimensional gel. Two-dimensional electrophoresis was performed as described (Cullen et al., 2002) . Duplicate gel sets for each of two independent sample preparations were used to perform image analysis as described . Protein spots showing a minimum twofold change in abundance were selected for characterization by mass spectrometry. MALDI-TOF MS peptide mass mapping was performed as described (Cullen et al., 2002) . Peptide masses were used to search the translated C. crescentus genome (http://www.tigr.org) using the programs PRO-TEINPROBE and MASSLYNX (Micromass).

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