Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, CA 90095-1569, USA
Correspondence
James W. Gober
gober{at}chem.ucla.edu
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ABSTRACT |
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Present address: Department of Genetics, Stanford University, Stanford, CA 94305, USA.
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INTRODUCTION |
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The expression of class III and IV flagellar genes requires RNA polymerase, containing the 54 subunit, and the transcriptional activator FlbD (Ramakrishnan & Newton, 1990
; Wingrove et al., 1993
; Benson et al., 1994a
, b
; Mullin et al., 1994
; Wu et al., 1995
; Muir & Gober, 2002
). FlbD contains a conserved amino-terminal, two-component, response-regulator domain, as well as a conserved, central ATPase domain and carboxyl-terminal DNA-binding domain characteristic of the family of
54 transcriptional activators (Ramakrishnan & Newton, 1990
). In addition to being subject to cell cycle-regulated phosphorylation (Wingrove et al., 1993
), FlbD activity is also influenced by the progression of flagellum assembly (Muir et al., 2001
; Muir & Gober, 2002
, 2004
). FlbD exhibits markedly reduced activity in mutant strains that do not assemble a class II-encoded basal-body structure. Gain-of-function mutations in FlbD that bypass the requirement for a class II flagellar structure have been isolated (Mangan et al., 1995
; Muir & Gober, 2002
). These mutant strains exhibit an aberrant temporal and spatial pattern of class III/IV transcription, highlighting the importance of this flagellar-assembly checkpoint in regulating cell-cycle transcription.
The status of flagellum assembly is transduced to FlbD by the trans-acting factor FliX (Muir et al., 2001; Muir & Gober, 2002
, 2004
). In one model, in the absence of a class II flagellar structure, FliX represses FlbD activity, and when a class II-encoded flagellar structure is completed, FliX activates FlbD. Genetic experiments have indeed shown that FliX functions as both a positive and negative regulator of FlbD activity (Muir et al., 2001
; Muir & Gober, 2002
, 2004
). Mutant strains containing a deletion in fliX are non-motile and fail to express class III and IV flagellar genes (Mohr et al., 1998
; Muir et al., 2001
). Gain-of-function mutations in flbD restore motility to fliX mutant strains, indicating that FliX functions in the same pathway as FlbD in the activation of late flagellar-gene transcription (Muir et al., 2001
; Muir & Gober, 2002
). Consistent with this idea, a gain-of-function mutant fliX that permits class III/IV flagellar-gene expression in the absence of a class II-encoded structure has also been isolated (Muir et al., 2001
). FliX has recently been shown to interact with FlbD in a bacterial two-hybrid assay, indicating that they may form a complex in Caulobacter cells (Muir & Gober, 2004
). Interestingly, FlbD and FliX are conserved in several species of alphaproteobacteria possessing polar flagella, thus constituting a regulatory pair in organisms that assemble flagella under cell-cycle control.
The FliX/FlbD regulatory pathway is also required for normal cell division (Muir & Gober, 2001). Strains containing mutations in class II flagellar-structural genes, flbD or fliX exhibit a cell-division defect characterized by the accumulation of filamentous cells in late exponential-phase cultures (Yu & Shapiro, 1992
; Gober et al., 1995
; Zhuang & Shapiro, 1995
; Muir & Gober, 2001
). These filamentous cells often contain multiple constrictions along the cell length, indicative of a late-stage cell-division defect. Experiments examining the effects of overexpressing dominant-negative alleles of the early flagellar gene flbE demonstrated that the filamentous phenotype in class II flagellar-mutant strains was attributable to an absence of FlbD activity (Muir & Gober, 2001
). The same gain-of-function mutations in flbD that restore late flagellar-gene transcription in class II flagellar mutants also completely ameliorated the cell-division defect in these mutant cells. Thus, the early flagellar-assembly checkpoint regulates both cell division and late flagellar-gene transcription by modulating FlbD activity. The simultaneous operation of these two checkpoints assures that progeny swarmer cells possess a fully functional flagellum at the time of cell division.
In this report, we examine the relationship between flagellum assembly, cell division and the FliX/FlbD regulatory pathway. We demonstrate that FliX-mediated negative regulation of FlbD activity is responsible for the cell-division defect in class II flagellar mutants. Additionally, we show that this effect is attributable to an absence of FlbD activity. By using a strain depleted of FtsZ, we also demonstrate that cell division is not required for the proper timing of the initiation of FliX/FlbD-dependent transcription of late flagellar genes. Prolonged inhibition of cell division resulted in the eventual loss of FlbD activity, which could be reversed by gain-of-function mutations in flbD, but not in fliX, indicating the existence of a second cell cycle-dependent pathway for FlbD activation.
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METHODS |
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Microscopy.
For the visualization of live cells, grown cultures were concentrated by harvesting the cells in a microcentrifuge, removing 90 % of the supernatant and suspending the cell pellet in the remainder of the supernatant. Cells (2 µl) from this suspension were mixed with 1 µl FM4-64 (25 µg ml1; Molecular Probes) and the sample was covered with a poly-L-lysine-treated coverslip. Samples were visualized by using fluorescence microscopy. Images were captured and analysed by using the Resolve3D image-acquisition software package (Applied Precision). In order to visualize cells by using transmission electron microscopy, late exponential-phase cultures were grown in PYE medium; the cells were then harvested, washed once in 150 mM NaCl and then suspended in 150 mM NaCl. This cell suspension was applied to carbon-coated grids; the cells were allowed to adhere and were then stained with 1 % uranyl acetate for approximately 45 s.
Visualization of the flagellum was done by using an immunofluorescence microscopy protocol adapted from that of Maddock & Shapiro (1993). YB1585 (ftsZ depletion) cells from synchronized cultures grown in the presence or absence of xylose to the late predivisional cell stage (starting culture OD600, 0·4) were fixed in a final concentration of 3·0 % formaldehyde and 30 mM sodium phosphate, pH 7·5, for 45 min at room temperature; 37 % formaldehyde and 1 M sodium phosphate were added directly to 1 ml culture. Fixed cells were harvested and rinsed three times with cold PBST (140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1·5 mM KH2PO4 and 0·05 % Tween 20) in 1·5 ml microfuge tubes. Pelleted cells were suspended in 200 µl cold PBST and placed onto poly-lysine-coated slides. Cells were left to adhere to slides at room temperature for 5 min. Non-adherent cells were removed by aspiration and those remaining on the slide were incubated at room temperature in ice-cold PBST containing 2 % BSA for 15 min. Slides were then washed once with cold PBST and incubated with a 1 : 200 dilution of anti-flagellin antisera in PBST in a humid chamber at room temperature for 2 h. Slides were rinsed twice with cold PBST and incubated for 1 h in the dark, under the conditions described above, with a secondary antibody conjugated to CY3 (Jackson Immunoresearch). Slides were then rinsed twice with cold PBST and dried by aspiration. Immediately after drying, a 510 µl drop of 10 % glycerol was placed onto the treated cells, followed by a coverslip. Slides were either visualized immediately or stored at 4 °C in the dark after edges of coverslips had been sealed with nail polish in order to prevent dehydration.
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RESULTS |
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When these isolated YB1585 swarmer cells were incubated in medium containing xylose and allowed to progress through the cell cycle, the fliKlacZ reporter fusion had a peak rate of expression in late predivisional cells that declined following cell division (Fig. 5a). This pattern of expression was similar to that of flagellin protein (Fig. 5b
), as well as that reported for fliK expression in wild-type cells of C. crescentus (Muir & Gober, 2002
). FtsZ levels were monitored in this same experiment via immunoblot with an anti-FtsZ antibody. As has been reported previously (Quardokus et al., 1996
; Kelly et al., 1998
), FtsZ protein was below detectable limits in isolated swarmer cells and returned in stalked cells, with peak levels appearing in predivisional cells (Fig. 5b
). We also assayed the effects of expressing the gain-of-function alleles of FliX (FliX1) and FlbD (FlbD-1204) on the timing of fliKlacZ expression in YB1585 (Fig. 5a
). In cells in which the fliX1 allele was the sole copy of fliX in the cell, the temporal pattern of fliKlacZ expression was almost identical to that of cells containing wild-type fliX (Fig. 5a
). In contrast, YB1585 cells containing the gain-of-function flbD-1204 allele exhibited aberrant timing of fliKlacZ expression. In addition to having high levels of expression in predivisional cells, this strain also expressed the fliKlacZ fusion late in the cell cycle and in swarmer cells (Fig. 5a
). This pattern of expression is similar to that reported previously for strains carrying this flbD-1204 allele in a wild-type genetic background (Muir & Gober, 2002
).
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These results show that FlbD does not require the earliest known step in the cell-division cycle in order to undergo cell cycle-regulated activation. Although they expressed late flagellar genes (Fig. 5b), the cells lacking FtsZ were non-motile (data not shown). This is consistent with previous observations (Terrana & Newton, 1976
; Huguenel & Newton, 1982
; Ohta et al., 1997
; Matroule et al., 2004
) demonstrating that an inhibition of cell division resulted in non-motile cells possessing a completely assembled flagellum. Likewise, immunofluorescence microscopy employing an anti-flagellin antibody to visualize the flagellar filament revealed the presence of an intact flagellar filament in the synchronized cells lacking FtsZ (Fig. 5c
).
Cell division is required for reactivation of FlbD following flagellum assembly
C. crescentus cells depleted of FtsZ continue to lengthen and replicate chromosomal DNA for what would represent several generations (data not shown). We wanted to observe the long-term, i.e. greater than one generation, effects of FtsZ depletion on FlbD activity. One hypothesis is that the persistent presence of an intact polar flagellum in FtsZ-depleted cells would eventually result in a repression of FlbD activity, as C. crescentus possesses regulatory mechanisms to shut off flagellar-gene expression following the completion of flagellum assembly (i.e. through the repressive effects of FliX) (Muir & Gober, 2002, 2004
). In order to test this idea, YB1585 cells containing a fliFlacZ reporter fusion were grown overnight in the presence of xylose, harvested, washed and suspended in fresh medium in either the presence or absence of xylose (Fig. 6a
).
-Galactosidase activity was assayed over time, up to what would represent approximately 2·5 generations (5 h). As the fliF promoter is repressed by FlbD (Benson et al., 1994a
; Mullin et al., 1994
; Wingrove & Gober, 1994
), an increase in
-galactosidase activity would be indicative of a loss of FlbD activity. Indeed, the gradual loss of FtsZ by this strain resulted in a loss of FlbD activity over time, as indicated by an increase in fliFlacZ promoter activity (Fig. 6c
). We next wanted to test the idea that the loss of FlbD activity was a consequence of the repressive effects of FliX. We assayed whether the loss of FtsZ would cause a decrease in FlbD activity in a strain bearing the gain-of-function fliX1 allele. Previous experiments have demonstrated that this allele of fliX is locked as a positive activator and cannot repress FlbD activity, even following successful flagellum assembly or in class II mutant strains (Muir & Gober, 2004
). Surprisingly, when cells carrying the fliX1 allele were depleted of FtsZ, there was also a loss of FlbD activity, as indicated by the increase in fliFlacZ promoter activity (Fig. 6d
). This result indicates that the loss of FlbD activity under conditions of FtsZ depletion is not related to the presence of an intact flagellum in these cells and the accompanying repressive actions of FliX. Next, we determined whether the flbD-1204 gain-of-function allele would also be subject to a loss of activity when cell division was inhibited. In this case, there was a gradual loss of fliFlacZ promoter activity over time following FtsZ depletion, indicating that FlbD-1204 activity slightly increased over the course of the experiment (Fig. 6e
).
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DISCUSSION |
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FliX and FlbD regulate the expression of late flagellar genes, encoding the distal rods and rings of the basal body, the hook (class III) and the filament (class IV). Previous experiments have shown that these genes are not transcribed in the absence of a class II-encoded flagellar structure (Newton et al., 1989; Xu et al., 1989
; Ramakrishnan et al., 1994
; Mangan et al., 1995
). FliX regulates the activity of the FlbD transcription factor in response to this early flagellar-assembly event, such that FlbD activity is repressed prior to flagellar assembly and activated via FliX action following completion of the early basal-body structure (Muir et al., 2001
; Muir & Gober, 2002
, 2004
). Therefore, the same regulatory pathway controls the transcription of late flagellar genes and the completion of cell division. We hypothesize that the filamentous phenotype of class II flagellar mutants is attributable to a regulatory cell-cycle checkpoint that couples the assembly of an MS ringswitchTTSS complex (class II structure) to the completion of cell division. The experiments presented here demonstrate that this checkpoint operates through the regulation of FliX and FlbD activity, as the presence of constitutive alleles of either trans-acting factor resulted in the formation of morphologically normal cells, even in the absence of a completed flagellar structure.
In E. coli, cell division initiates with the formation of a polymerized ring of FtsZ at the midcell (Bi & Lutkenhaus, 1991), followed by the recruitment of other early-acting cell-division proteins including, among others, FtsA, FtsQ, FtsI, FtsW, FtsN and ZipA (reviewed by Lutkenhaus & Addinall, 1997
). The assembly of this complex at the midcell leads to a redirecting of peptidoglycan assembly with the eventual formation of a septum, followed ultimately by cell separation. In cultures of Caulobacter class II mutants, the cells often possess multiple constrictions along the cell length, indicative of an arrest in a late stage of cell division. This arrest is only partial, as these mutant cultures achieve a final cell density similar to that of wild-type cells. Therefore, the separation of class II-mutant cells is delayed with respect to cell elongation, resulting in an accumulation of filamentous cells with constrictions. Many of the cells that we observed by using light microscopy were similar in appearance to those expressing dominant-negative alleles of FtsZ (Wang et al., 2001
), possessing elongated constrictions along the length of the filament. Electron microscopy reveals that these predivisional cells very often appear to possess two completely separated compartments that are connected by a narrow bridge of material with a relatively electron-dense rod traversing the length of the structure and apparently connecting the two nascent compartments (Fig. 3
). We were unable to detect this unusual structure in predivisional cells from wild-type cultures. As Caulobacter cells pinch gradually at the midcell during cytokinesis, rather than septating (Poindexter & Hagenzieker, 1981
), this narrow neck of cell envelope-derived material in class II mutants probably arises from a delay in the constriction process. Specifically, the presence of the structure connecting the two nascent compartments suggests that the final stage in the constriction process, cell separation, is arrested. It is also possible that the presence of this structure is indicative of an aberrant cell-division pathway in class II flagellar mutants.
The completion of cell division and the expression of late flagellar genes parallel each other, with both processes being regulated by a common mechanism. We hypothesize that these checkpoints exist in order to ensure that each progeny swarmer cell possesses a fully functional flagellum. Thus, the completion of cell division is delayed until an early MS ringswitchTTSS complex is assembled, resulting in the activation of FliX and FlbD. FlbD, in turn, activates the transcription of late flagellar genes and, we speculate, a late cell-division gene. Motility is an essential aspect of the Caulobacter lifestyle: every sessile reproductive stalked cell gives rise to a temporarily non-reproductive swarmer cell. C. crescentus is an oligotrophic bacterium, often occupying nutrient-poor, freshwater environments (Poindexter, 1964). The obligatory asymmetrical cell division of this organism serves to disperse the nascent daughter cells via motility and chemotaxis. The existence of a checkpoint locking the completion of cell division and flagellar synthesis in step with one another underscores the importance of functional motility in the progeny swarmer cells.
We also examined the influence of cell division on the cell cycle-regulated activation of FlbD. The initiation of DNA replication has been shown to be a critical event in the activation of early flagellar-gene expression (Dingwall et al., 1992; Stephens & Shapiro, 1993
). This cell-cycle event is coupled to early flagellar-gene expression through the timed synthesis and phosphorylation of the global transcription factor CtrA (Quon et al., 1996
; Domian et al., 1997
; Reisenauer et al., 1999
). In addition to activating the transcription of early flagellar genes, CtrA also regulates as many as 95 genes directly (Laub et al., 2002
), including the early cell-division genes ftsZ (Kelly et al., 1998
) and ftsQA (Wortinger et al., 2000
). As early flagellar assembly and early cell division shared a common regulator, we examined whether initiation of cell division, like class II flagellar assembly, influenced the expression of late flagellar genes. We conducted these cell-cycle experiments in synchronized populations of cells that could not synthesize FtsZ and found that the initiation of cell division was not required for regulating the temporal activation of FlbD. Interestingly, subsequent cell division was required for the normal cessation of class III transcription in these experiments. FlbD, however, did not remain active for longer than what would represent one generation in these FtsZ-depleted cells. FlbD activity was lost gradually in FtsZ-depleted cells that did not divide. One possibility was that the presence of the completed flagellum was inhibiting FlbD activity via the action of FliX (i.e. one function of FliX is to shut off FlbD activity following the completion of flagellar assembly). Surprisingly, we found that the loss of FlbD activity was not attributable to FliX-mediated repression, as the presence of a constitutively active allele of fliX (fliX1) could not rescue FlbD activity under conditions of FtsZ depletion. However, there was no measurable loss of FlbD activity when the constitutive flbD-1204 allele was expressed under these conditions. These results suggest that FlbD is activated by a second, distinct, cell cycle-regulated mechanism and that this regulatory action is required for its ensuing activation by early flagellar assembly via FliX activity. The data also indicate that this cell cycle-related activation of FlbD requires cell division, followed perhaps by a round of DNA replication. One possibility is that FlbD becomes unphosphorylated upon FtsZ depletion, and requires cell division in order to be rephosphorylated. Indeed, previous experiments have indicated that FlbD is phosphorylated in a cell cycle-regulated fashion (Wingrove et al., 1993
). Identification of the kinase(s) that phosphorylate FlbD will resolve whether this activity is regulated by cell division.
The depletion of FtsZ from synchronized cultures led to the synthesis of an intact, and apparently complete, flagellum that did not rotate. Therefore, early flagellar assembly regulates cytokinesis, which in turn regulates the acquisition of motility by the fully assembled flagellum. The results presented here suggest that FliX and FlbD have a central role in helping to orchestrate this ordered sequence of developmental checkpoints.
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ACKNOWLEDGEMENTS |
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Received 5 May 2005;
revised 12 August 2005;
accepted 15 August 2005.
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