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Genetics Program, Tufts University School of Medicine, and Howard Hughes Medical Institute, Boston, Massachusetts 02111, USA
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[Keywords: Vibrio cholerae; chromosome segregation; ParA; ParB]
Received September 25, 2006; revised version accepted October 18, 2006.
; Webb et al. 1997), and the relative spatial arrangement of most chromosomal loci correlates with their linear map positions (Teleman et al. 1998; Niki et al. 2000; Viollier et al. 2004). Specific chromosomal regions, particularly those close to the origin of replication, are actively and rapidly positioned in the cell (Glaser et al. 1997; Webb et al. 1998; Jensen and Shapiro 1999). The observed movements are more rapid than could be accounted for by previous models of passive segregation mediated by chromosome attachment to the growing cell membrane (Jacob et al. 1963).
Several mechanisms may contribute to the dynamic movements of bacterial chromosomes (for reviews, see Errington et al. 2005; Leonard et al. 2005). The observation in Bacillus subtilis that the DNA replication machinery appears to be localized as a stationary "factory" at the cell center (Lemon and Grossman 1998) led to the proposal that DNA polymerase could provide the bidirectional force for segregation (Lemon and Grossman 2000). Similarly, RNA polymerase, acting on directionally biased genes near the origin, has been hypothesized to impart both motive force and directionality to segregation (Dworkin and Losick 2002; Kruse et al. 2006). While both of these models could potentially explain the symmetric and bidirectional segregation of the Escherichia coli and B. subtilis chromosomes from the middle of the cell, it is difficult to directly apply them to the asymmetric segregation process that occurs in Caulobacter crescentus (Mohl and Gober 1997; Viollier et al. 2004) and in Vibrio cholerae (Fogel and Waldor 2005). In both of these organisms, the origin region is located close to one pole (the "old" pole) early in the cell cycle, and after replication, one copy remains at that pole while the other traverses the entire length of the cell to the opposite ("new") pole. Consistent with an asymmetric segregation pattern from the pole, the replication machinery of C. crescentus localizes to the old pole, and then after replication initiation, migrates to the cell center (Jensen et al. 2001). The presence of a moving replisome suggested a modification of previous models in which the origin regions are positioned rapidly by an origin-specific mechanism, and then the bulk of the chromosome is segregated by a replication factory in directions established by the positioning of the origins at the poles (Jensen et al. 2001).
Most bacterial chromosomes encode orthologs of plasmid partitioning (Par) proteins near their origins (Gerdes et al. 2000). In plasmids, par loci consist of three components: a DNA-binding protein (often termed ParB), an ATPase (ParA), and a centromere-like site (parS). ParB binds parS and spreads along the DNA, forming a large nucleoprotein complex. Formation of this complex and its interaction with ParA are required for efficient plasmid segregation (Ebersbach and Gerdes 2005; Leonard et al. 2005). The Par-family ATPases fall into two distinct phylogenetic groups; type I ParAs contain the conserved Walker-box ATP-binding motif, whereas type II ParAs are structurally related to eukaryotic actin (Gerdes et al. 2000). Types I and II ParAs are found in different plasmid families, but only type I par loci have been identified on bacterial chromosomes (Gerdes et al. 2000). Both type I and type II ParAs form ATP-dependent filamentous polymers in vitro (Møller-Jensen et al. 2002; Barillà et al. 2005; Lim et al. 2005). Type II ParAs appear to mediate plasmid segregation by polymerizing between plasmid pairs and "pushing" them apart toward the poles (Møller-Jensen et al. 2003). The mechanism by which type I plasmid ParAs function is less clear. Type I ParAs from some plasmids appear to oscillate back and forth in the cell (Ebersbach and Gerdes 2001; Lim et al. 2005; Adachi et al. 2006), but it is unknown how oscillation positions plasmids. Recently, a plasmid ParA was shown to polymerize into radial filaments on ParB-bound DNA in vitro, and a model was proposed in which plasmids are positioned by ParA pushing in all directions in the cell (Lim et al. 2005).
While the essential role of par loci in plasmid partitioning has been long appreciated, their functions in bacterial chromosome biology is less clear. The B. subtilis Par proteins Soj (ParA) and Spo0J (ParB) are nonessential but have effects on chromosome segregation (Ireton et al. 1994; Sharpe and Errington 1996; Lee et al. 2003; Wu and Errington 2003; Lee and Grossman 2006). Spo0J binds to at least 8 sites in a large region around the origin (Lin and Grossman 1998), and deletion of spo0J results in an increased frequency of anucleate cells (Ireton et al. 1994). Together, Spo0J and Soj appear to facilitate efficient separation of newly duplicated origins (Lee and Grossman 2006), but a mechanistic understanding of their role in chromosome segregation remains to be defined. In contrast to B. subtilis, the C. crescentus ParA and ParB are essential, and their overexpression or depletion results in defects in cell growth, division, and chromosome segregation (Mohl and Gober 1997; Mohl et al. 2001). ParB of C. crescentus binds to sites near the origin of the chromosome (Mohl et al. 2001) and localizes as foci at the extreme poles (Mohl and Gober 1997). While C. crescentus ParA and ParB affect both cell division and chromosome segregation, recent evidence suggests that the cell division defects are due to the effects of chromosome segregation on the localization of MipZ, an inhibitor of FtsZ ring formation (Thanbichler and Shapiro 2006). Thus, while there is strong evidence in several bacteria for active movement of chromosomal DNA during segregation, specific molecular mechanisms that mediate such movements have not been clearly elucidated.
V. cholerae is a curved, Gram-negative rod that causes the severe diarrheal disease cholera. It contains two circular chromosomes, 2.96 and 1.07 Mb (chrI and chrII, respectively). Despite having coordinated replication initiation (Egan et al. 2004), the segregation of the two chromosomes is likely governed by distinct mechanisms as the localization and segregation patterns of their origin regions are different (Fogel and Waldor 2005; Fiebig et al. 2006). The origin region of chrI is localized near the old pole, where an asymmetric segregation process is initiated in which the duplicated origin segregates across the cell to the new pole. In contrast, the origin of chrII is localized at the cell center, where after replication, and at a later point in the cell cycle, the duplicated origins move symmetrically to the quarter positions of the cell, which become the new cell centers.
The distinct localization and segregation dynamics of the origin regions of the two chromosomes suggests the existence of mechanisms capable of discriminating between and interacting with the two origin regions to mediate their cell cycle-dependent segregation dynamics. Both chromosomes contain unique origin-proximal parAB loci. Here we explored the role of the Par proteins in the segregation of chrI. Our findings suggest that the chrI Par proteins participate in a mitotic-like pulling mechanism to mediate the asymmetric polar segregation of the origin region of chrI in V. cholerae.
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Fusions of GFP to ParB proteins in several plasmid and chromosomal systems form punctate fluorescent foci, marking the location of the ParB–parS nucleoprotein complex in the cell (Glaser et al. 1997; Lin et al. 1997; Li and Austin 2002). We constructed a fusion of YFP to the N terminus of the ParB protein encoded on chrI (ParBI) in order to investigate if its subcellular localization was suggestive of a role in chromosome segregation. YFP-ParBI formed well-defined fluorescent foci in wild-type V. cholerae cells (Fig. 1A). In contrast, expression of this fusion protein in E. coli resulted in only diffuse fluorescence (Supplementary Fig. S1), suggesting that there are V. cholerae specific binding sites for ParBI. Almost all V. cholerae cells with YFP-ParBI foci contained at least one focus at the extreme pole of the cell (94%). The majority of cells (67%) contained two foci, one at the extreme pole and the second either at the opposite pole or at an intermediate position between the poles (Fig. 1B). The number of foci per cell and their localization was very similar to our observations of the origin of chrI (oriCIvc) detected with the lacO/tetO fluorescent repressor–operator systems (FROS) (Fogel and Waldor 2005), suggesting that ParBI likely binds near the origin of chrI.
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defined the binding site for the B. subtilis ParB ortholog Spo0J and noted the widespread conservation of this sequence in many bacteria. In V. cholerae, there are three copies of this sequence on chrI and none on chrII. The three sites are clustered together 
65 kb to the right of oriCIvc, with 3 kb separating them. We found that these three sites, and not closely related sequences elsewhere on chrI or chrII, formed foci with YFP-ParBI when tested in E. coli (Supplementary Fig. S1; data not shown). As many newborn V. cholerae cells contained only a single focus (Fig. 1B, top), and the number and distribution of foci were qualitatively similar to those of origins labeled with other markers (Fogel and Waldor 2005; Fiebig et al. 2006), we concluded that there is likely one YFP-ParBI focus per copy of oriCIvc, marking the subcellular location of the origin-proximal parSI locus. Expression of YFP-ParBI for the time periods used in these experiments did not affect the growth rate of wild-type cells, nor does it alter the localization of other origin regions visualized by FROS (data not shown).
Analysis of foci in more than a thousand cells revealed that YFP-ParBI foci are often found at the extreme pole (Fig. 1C,D). In cells with two YFP-ParBI foci, the focus closest to a pole (Fig. 1C, blue circles) was on average only 4.3% of the cell length away, and many foci overlapped the edge of the cell. The other focus (Fig. 1C, orange squares) was found close to the opposite pole or at intermediate positions between the poles, presumably representing an actively segregating origin. As expected, time-lapse microscopy (Fig. 1E) revealed that the YFP-ParBI foci segregate with asymmetric dynamics that recapitulate the segregation pattern of the FROS-labeled origins (Fogel and Waldor 2005). Late predivisional cells contain YFP-ParBI foci at both poles; these poles become the old poles of each daughter cell. Around the time of division, the polar foci duplicate, and one segregates across the cell to the new pole; the other remains at the old pole. The most significant difference between YFP-ParBI foci and the origin-proximal loci studied previously with FROS is the extreme polarity of YFP-ParBI; the FROS foci were rarely at the edge of the cell. The extreme polarity of the YFP-ParBI foci suggested the possibility that the ParBI–parSI nucleoprotein complex anchors the chromosome to the pole, as has been proposed for C. crescentus (Mohl and Gober 1997). The origin, 65 kb away, would be expected to be near the pole, but not as close as the anchored site.
ParAI is required for polar positioning and asymmetric segregation of ParBI
We constructed a V. cholerae strain with a deletion of parAI. The 
parAI strain had an increased frequency of filamentous cells, 1.8% versus 0.4% for wild type, suggesting a mild defect in cell division, but otherwise the strain appeared to grow normally. We examined the localization of YFP-ParBI in the parAI mutant and found that the foci were dramatically mislocalized (Fig. 1F). In parAI cells, the YFP-ParBI foci were dissociated from the cell poles and instead were generally found near the cell center in cells with a single focus or close to the quarter positions in cells with two foci (Fig. 1G). In parAI cells with two foci, the mean position of the closest-to-pole ParBI focus (Fig. 1H, blue circles) was 16.4% of the cell length (vs. 4.3% for wild type), and >98.5% of the foci were farther away from the pole than the mean value for wild type. Not only was the mean position of the closest-to-pole focus changed, but the distribution of the distances was significantly wider for the parAI strain (Fig. 1, cf. D and I). This suggests that YFP-ParBI foci in the parAI strain have a greater degree of freedom in their localization, a behavior that is consistent with loss of an attachment between the ParBI-bound chromosome and the pole. Recently, mislocalization of an origin-proximal locus in a V. cholerae parAI deletion mutant was shown by FISH (Saint-Dic et al. 2006). Together these observations suggest that ParAI affects the localization of a relatively large origin-proximal domain of chromosome I.
Remarkably, the asymmetric pattern that characterizes the segregation of oriCIvc was absent in the parAI mutant. Time-lapse analysis of parAI cells showed generally bidirectional movement of YFP-ParBI foci (Fig. 1J). The presence of separated and symmetrically distributed YFP-ParBI foci in the parAI strain (Fig. 1G,J) suggests that ParAI-independent mechanisms can still segregate duplicated origins, even though wild-type polar localization and asymmetric segregation are lost.
The defects in the parAI cells are attributable to the deletion of parAI, as expression of either a His-tagged ParAI (ParAI-His) or a fluorescent fusion to ParAI (ParAI-CFP) restored the polar localization of YFP-ParBI foci (Fig. 2A,B, respectively). Moreover, the ability of ParAI to localize ParBI to the pole likely requires ATP, as substitutions in the conserved ATP-binding motif of ParAI abolished the ability of ParAI-CFP to complement the parAI deletion (Fig. 2C,D). In addition to the parSI site, other origin-proximal loci on both sides of oriCIvc were also mislocalized in the parAI mutant (see below). These observations suggest that ParAI is required to position the ParBI–parSI complex at the extreme pole, thereby affecting the localization of a relatively large origin-proximal region.
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In order to study the spatial relationship between parSI and another origin-proximal locus, we expressed CFP-ParBI and TetR-YFP in a strain containing a tetO array integrated 15 kb to the right of oriCIvc [Fig. 3A, tetO(2)]. Under the growth conditions used, the majority (85%) of these dual-labeled cells had replicated the origin region of chrI and therefore had four foci, one CFP-ParBI and one TetR-YFP for each copy of the origin region (Fig. 3B). As expected from their localization in single fluorescence experiments (Fig. 1; Fogel and Waldor 2005), most cells contained one CFP-ParBI and one TetR-YFP focus at the extrema of one pole, and a second pair either at the opposite pole or at intermediate positions (Fig. 3B). In most cells, for the pair of foci closest to a pole (the old pole), the CFP-ParBI and TetR-YFP foci were adjacent or overlapping, as might be expected for two loci separated by only 50 kb (Fig. 3B, single white star). Despite this close association, their orientation with respect to the pole was not random. In fact, for 95.2% of cells (n = 849), the CFP-ParBI foci were found to be closer to the pole than the TetR-YFP foci (Fig. 3B, single white star; Fig. 3C). Analysis of parSI and a tetO-tagged locus on the other side of the origin [Fig. 3A, tetO(1)] similarly showed that parSI was consistently closest to the pole (data not shown). The ordered arrangement of these foci both relative to each other and to a cellular structure (the pole) reveals an extremely fine level of spatial organization for this region of the chromosome.
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In contrast to the close proximity of CFP-ParBI and TetR-YFP foci at the old pole, for the segregating foci, CFP-ParBI was often dramatically separated from its companion TetR-YFP focus (Fig. 3B, white double arrow). The interfocal distances were often >1 µm and in some cells as much as 1.6 µm (Fig. 3D orange bars), as if strong forces were stretching the two foci apart. To our knowledge, such remarkable dynamic variability in the distance between two close genetic loci has not been reported before in bacteria. Similar behavior has been observed in eukaryotic cells between sites in the centromere and nearby sites on the chromosome arms (Goshima and Yanagida 2000). In this case, the separation was attributed to the action of mitotic spindles pulling at the centromere. In addition, the segregating YFP-ParBI foci, but not those at the pole, were often significantly elongated (Fig. 3B, double white star) as if subject to a distorting force. This distortion, together with the ordered arrangement and dynamic separation of parSI relative to adjacent loci, is consistent with the parSI site serving as the target for active segregation machinery.
When parSI (CFP-ParBI foci) and tetO(2) (TetR-YFP foci) were visualized together in the parAI background, as expected, both were significantly mislocalized (Fig. 3E,F). In cells with two foci, the position of the closest-to-pole CFP-ParBI focus as a fraction of cell length was 19.7% versus only 2.9% in the wild-type background, and for the TetR-YFP foci, 19.2% versus 9.4% (Fig. 3, cf. C and F). In addition to their mislocalization, the ordered arrangement (parSI always closer than tetO2) relative to the pole was not observed; instead, their orientation appeared random (Fig. 3, cf. B and E). Furthermore, the interfocal distances between CFP-ParBI and TetR-YFP foci were markedly reduced in the absence of ParAI. In parAI cells containing two pairs of foci, there were no cells in which CFP-ParBI and TetR-YFP foci were separated by >0.6 µm (Fig. 3D, blue bars) and the mean interfocal distance was only 0.19 µm compared with a mean of 0.65 µm in the wild-type strain. Thus, ParAI is required for a cell cycle-specific (i.e., during segregation) separation of the 50 kb of DNA between parSI and tetO(2). This requirement is consistent with a role for ParAI in applying force during segregation. The loss of the ordered arrangement relative to the pole suggests that ParAI continues to exert effects on the origin region both before and after segregation.
ParAI has a dynamic and complexsubcellular distribution
To study how ParAI might act to affect origin dynamics, we examined the subcellular localization of fluorescent ParAI fusions able to complement the parAI deletion (see above, Fig. 2B). The distribution of ParAI-CFP in parAI cells generally conformed to one of three patterns roughly correlated with cell size (Fig. 4A). In smaller (younger) cells, there was invariably a distinct focus at one pole and a dense patch of fluorescence extending from the other pole (Fig. 4A, panels I). In early predivisional cells, the distribution of ParAI-CFP was more variable; many cells had foci, usually of different intensity, at both poles and, in general, little or no fluorescence throughout the cell body (Fig. 4A panels II). In the oldest cells, particularly those that had visibly initiated septation, the ParAI-CFP distribution was again highly consistent; dense bands or patches extended from the midcell or septum toward, but not reaching the poles, which were occupied by distinct foci (Fig. 4A panels III). The localization of ParAI fluorescent fusions was essentially the same in wild-type cells; in contrast, YFP or CFP alone produced only diffuse fluorescence (data not shown).
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parAI cells shows more clearly that ParAI-CFP does not fill the entire diameter of the cell (Fig. 4B) and suggests that it may form a cytoskeletal structure as fluorescence from two sections 0.1 µm apart do not completely colocalize (Fig. 4B merged). The same deconvolution processing on a z-stack of cells expressing the nonfunctional ParAI-CFP[K16E] showed no such structures (Fig. 4B).
Time-lapse experiments revealed that the localization of ParAI-CFP was highly dynamic. Although there was some variability, analysis of a large number of time-lapse experiments revealed consistent patterns, the most common of which is represented in Figure 4C. When a predivisional cell (Fig. 4C, time = 0) divides (time = 3), it produces two "newborn" cells both with the ParAI-CFP distribution seen in small cells (Fig. 4A panels I). Over a period of 20–30 min, the band of ParAI-CFP shrinks toward the new pole until the cell has only punctate foci at both poles. These dynamics, which occur only once per cell cycle, are different from what has been observed for some plasmid type I ParA proteins that have been shown to oscillate rapidly across the cell, redistributing from one half of the cell to the other every few minutes (Ebersbach and Gerdes 2001; Lim et al. 2005). Interestingly, the ParA ortholog on chromosome II (ParAII), a type I ATPase more related to plasmid ParA proteins, oscillates rapidly (Supplementary Fig. S2). Our observations suggest that the V. cholerae ParAI has different behavior that involves cell cycle-coordinated retraction toward the new pole.
YFP-ParBI and ParAI-CFP colocalize,and their dynamics are coordinated
To explore the relationship between ParAI and ParBI, YFP-ParBI and ParAI-CFP were coexpressed in parAI cells. These experiments revealed two different types of association: (1) Polar YFP-ParBI foci colocalized with polar ParAI-CFP foci (Fig. 5A, white arrowhead); and (2) in cells with two YFP-ParBI foci, the segregating focus was always at the edge of a band of ParAI-CFP fluorescence (Fig. 5A, white star). In small cells containing two close, presumably recently separated, YFP-ParBI foci (Fig. 5B panels I,II), the band of ParAI-CFP extended across the cell from the new pole to the segregating YFP-ParBI focus. In older cells, in which the segregating YFP-ParBI focus was almost at the opposite pole, the band of ParAI-CFP was smaller, filling only the space between the segregating YFP-ParBI focus and the new pole (Fig. 5B, panels III,IV). In late predivisional cells (Fig. 5B panels V,VI), YFP-ParBI foci are at each pole, and ParAI-CFP was almost completely located at the cell center or septum from which it extended outward toward the two old poles. Thus, before division is complete, both ParAI and ParBI are positioned to repeat the cycle.
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Mutations in the ATP-binding motif of ParAI abolish its dynamics
As the activity of many Walker-type ParA proteins has been shown to require ATP hydrolysis (Leonard et al. 2005), we made two separate substitution mutations, K16E and K16Q, in the predicted ATP-binding pocket of the ParAI-CFP fusion protein. In a plasmid ortholog of ParAI, these substitutions have been reported to block ATP binding (K16E) and to allow binding but prevent hydrolysis (K16Q), respectively (Fung et al. 2001). As described above, neither construct was capable of complementing the mislocalization of YFP-ParBI foci in the parAI background (Fig. 2C,D). We examined the subcellular distribution of these constructs to investigate the effects of these mutations on ParAI dynamics. In both wild-type and parAI backgrounds, ParAI-CFP[K16E] showed only homogeneous diffuse fluorescence (Fig. 6A) and had no detectable dynamics in time-lapse experiments (data not shown). The K16Q mutation similarly resulted in diffuse fluorescence and lack of wild-type localization and dynamics; however, it also formed faint foci (Fig. 6A). When YFP-ParBI was coexpressed with ParA-CFP[K16Q], their foci were always colocalized (Fig. 6B). The loss of ParAI dynamics with both substitutions suggests that ATP binding and hydrolysis are required for the dynamic localization of ParAI. The colocalization of the K16Q mutant, but not the K16E mutant, with YFP-ParBI foci suggests that the ATP-bound state, but not the unbound state (presumably the case for the K16E substitution), may promote the interaction of ParAI with ParBI.
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), it is likely that the Par-directed pulling segregation mechanism described here is conserved in many other systems.
ParBI localizes with parSI sites near the origin
The formation of visible foci by fluorescent ParBI fusions suggests that, like other ParB proteins, ParBI forms a large nucleoprotein complex on chrI DNA. In comparison to the distribution of the eight or more Spo0J-binding sites over a 800-kb region that spans the origin of the B. subtilis chromosome (Lin and Grossman 1998), the three parSI sites on V. cholerae chrI are much closer together. These conserved ParB-binding sites, originally identified in the V. cholerae genome by Lin and Grossman (1998), were recently shown to be functional for ParABI-mediated plasmid stabilization (Saint-Dic et al. 2006). The greater number and distribution of parS sites in B. subtilis may reflect an adaptation of the Par origin-localizing system to facilitate large-scale compaction and localization of the chromosome during sporulation. As in V. cholerae, the four to five parS sites in C. crescentus are clustered together within an 10-kb region, close to the origin (Mohl et al. 2001). The similarity of the distributions of the V. cholerae and C. crescentus parS sites along with the polar localization of their respective origins suggest that the C. crescentus and V. cholerae chrI par systems likely carry out the same function for origin localization, although they may have other roles that differ.
ParAI does not influence bulk nucleoid segregation
The ParABSI system primarily functions to position and segregate the origin region of chrI. Deletion of parAI did not dramatically disrupt partitioning of chrI to daughter cells, supporting the emerging view that bacterial chromosome segregation is mediated by multiple, likely overlapping or redundant mechanisms (Errington et al. 2005). Similarly, in C. crescentus, it was shown that MreB is required for proper positioning of the origin but not for other regions of the chromosome (Gitai et al. 2005). As C. crescentus origin localization is so similar to that of V. cholerae, it is possible that MreB plays similar roles in both bacteria, either as a separate mechanism or perhaps as a scaffolding for Par-mediated segregation, as proposed by Gitai et al. (2005). It seems likely that bulk chromosome segregation may be directed by conserved processes such as replication, transcription, and condensation; perhaps additional, species-specific, mechanisms might mediate the localization of specific chromosome regions such as the origin and terminus.
The parAI mutant has only a subtle growth defect despite the dramatic mislocalization of origin proximal regions in this background. The function of the extreme polar localization of chromosomal origins in bacteria such as V. cholerae and C. crescentus is unclear. Perhaps there are particular environmental conditions in which there is a strong advantage to having a more elongated and/or organized genome. If the chrI par genes do influence the segregation of origin-distal regions of the chromosome, then redundant mechanisms must be able to compensate in their absence. It will be interesting to examine if mutations in other genes implicated in chromosome segregation, such as mreB and mukB, have synthetic effects with parAI, as has been recently reported for soj in B. subtilis (Lee and Grossman 2006).
It is possible that there is cross-talk between the chrI and chrII par systems; the absence of a significant growth defect in the parAI mutant may be attributable to the activity of ParAII, encoded in the par locus on chrII. While ParAII is phylogenetically grouped with plasmid ParA proteins, both are type I ATPases, and they are 45% similar. Consistent with this idea, the localization and segregation of the ParB–parSI complex in the parAI background resembles the dynamics of oriCIIvc. While the mechanism to properly localize the parSI site at the pole is clearly dependent on ParAI, it is possible that ParAII might substitute for ParAI in other important steps during segregation. One interesting possibility is that the ParABI–parSI system may have a function specifically in origin separation, as is the case for Soj in B. subtilis (Lee and Grossman 2006). If so, ParAII might promote the separation of duplicated ParBI–parSI complexes, thus partially suppressing the parAI deletion.
Pulling or pushing DNA
Polymerizing ParA proteins could localize DNA by providing either "pushing" or "pulling" motive force (Møller-Jensen et al. 2002; Barillà et al. 2005). To date, the only well-established mechanism for Par-mediated DNA segregation is the ParM–ParR system of plasmid R1. ParM (a type II ParA ATPase) polymerizes bidirectionally between ParR-bound (ParB-like) plasmids. In this system, the ParM–ParR interaction stabilizes the ParM filament, facilitating its continued polymerization, thereby pushing the plasmid clusters in opposite directions in the cell (Møller-Jensen et al. 2002, 2003; Garner et al. 2004). Recently, another elegant mechanism was proposed for SopA/SopB-mediated segregation of the F plasmid that also invokes a pushing force. In this case, radial asters of SopA filaments pushing in all directions were hypothesized to mediate F segregation (Lim et al. 2005). In both models, the polymerizing ParA-like protein is found between segregating ParB–DNA complexes. Therefore, the region of ParA fluorescence increases with the distance between ParB complexes. This is not the case with the V. cholerae ParAI and ParBI proteins; rather, ParAI is found between the segregating ParBI focus and the new pole; fluorescent ParAI "shrinks" toward the new pole with increased ParBI segregation, consistent with a pulling mechanism.
Several recent observations regarding the biochemistry of ParA proteins and of ParA–ParB interactions suggest how ParAI may pull the ParBI–parSI complex toward the new pole. In vitro, in the presence of ATP, ParA monomers can polymerize and form filamentous structures (Møller-Jensen et al. 2002, 2003; Barillà et al. 2005). The nature of the structure of ParAI in V. cholerae requires future investigation; many of our images raise the possibility that this structure is composed of many ParAI polymers, possibly even a network or lattice of small ParAI polymers. In some plasmid systems, ParA binding to ParB stimulates ParA hydrolysis of ATP, promoting its disassociation from ParB and depolymerization. Assuming that similar processes occur in vivo with the V. cholerae ParAI and ParBI proteins, then the interaction of ParAI filaments with the ParBI–parSI complex would result in their depolymerization, thereby pulling the origin region of chrI toward the new pole via a "Brownian ratchet" mechanism (Raj and Peskin 2006). In this type of mechanism, ParBI, present as part of a large ParBI/parSI complex, would interact with ATP-bound ParAI molecules at the ends of ParAI polymers (within a larger filamentous structure) and in so doing, stimulate their ATPase activity. ATP hydrolysis by the terminal subunit would result in its dissociation as a monomer of ParAI • ADP (i.e., depolymerization). If the ParBI–parSI complex is only free to diffuse toward the polymer (it would be prevented from diffusing away by simultaneous contact with multiple ParAI polymers), it will come into contact with the new leading edge of the ParAI polymer, and again experience a positive interaction that can be propagated along the DNA, shifting the average position of nearby regions closer to the polymer edge. The observed retraction of ParAI-CFP as YFP-ParBI segregates (Fig. 6) is consistent with the idea that ParBI stimulates depolymerization of ParAI polymers. As ParAI-CFP fluorescence is consistently associated with the nonsegregating YFP-ParBI foci at the old pole, it is possible that ParAI is undergoing a constant cycle of polymerization and depolymerization to anchor ParBI–parSI at the pole. This type of equilibrium might involve an unknown polar protein that stimulates ParAI polymerization, whereas ParBI–parSI antagonizes it. While the biochemistry of this interaction is still highly speculative, it is consistent with our observations regarding substitutions in ParAI, as a mutation likely to prevent ParAI ATP binding abolished ParAI–ParBI colocalization, whereas a different substitution predicted to allow ATP binding, but not ATP hydrolysis, colocalized with ParBI. Further studies of the biochemistry of these proteins will be important to understand the details of ParAI dynamics and how ParAI–ParBI interactions could generate force for chromosome movement.
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A list of the strains and plasmids used in this workis available in Supplementary Table S1. All V. cholerae strains used in this study were derived from the sequenced clinical isolate N16961 (Heidelberg et al. 2000). The parAI mutation was made by allele exchange, as described (Fogel and Waldor 2005), using pMF158, a derivative of pCVD442 (Donnenberg and Kaper 1991) containing homology with regions flanking parAI. The deletion was confirmed by PCR. yfp-parBI was constructed by "splicing by overlap extension" PCR (Horton et al. 1989) and then inserted into pBAD33 and pBAD18 (Guzman et al. 1995) to make plasmids pMF302 and pMF341, respectively. parAI-yfp and parAI-cfp fusions were constructed by inserting parAI upstream of the yfp or cfp gene of plasmids p4414 and p4416, containing monomeric versions of yfp and cfp (generously provided by S. Bunnell, Tufts Medical School, Boston, MA). After determining that the sequences of the resulting fusions were correct, they were subcloned into pBAD33, yielding pMF320 and pMF321. ParAI K16E (pMF322) and K16Q (pMF323) substitutions were introduced into these plasmids by PCR mutagenesis using the QuickChange kit protocol (Stratagene) and verified by sequencing. pMF310 was constructed by inserting tetR-yfp, derived from pLAU53 (Lau et al. 2003) downstream from cfp-parBI in plasmid pMF303 (pBAD33-cfp-parBI). Strains YBB025 and MF302, which harbor tet operators inserted in vc0018, were constructed using allele exchange vector pYBA016, a pCVD442 derivative with the tetO cassette from pLAU44 (Lau et al. 2003) ligated into the XbaI site of a fragment of vc0018.
Microscopy
Cells were routinely prepared for microscopy by inoculating fresh single colonies taken from LB plates into M63 minimal media containing 0.1% casamino acids and 0.2% glucose and grown at 37°C to a density of 0.3–0.5 OD600 units. Expression of the fluorescent fusions from pBAD plasmids was induced by addition of 0.08% arabinose for 20–30 min. For experiments with strains containing the tetO cassette, 80 nM Anhydrotetracycline was added to the culture media at the same time as arabinose, to reduce TetR-YFP binding as described previously (Lau et al. 2003). Ten microliters to 20 µL of the broth culture was adsorbed onto thin agarose pads on microscope slides and allowed to settle for 3–4 min; then, the remaining media was aspirated and a coverslip were placed on top. Slides were sealed with nail polish. Images were acquired with a Zeiss Axioplane 2 microscope equipped with a 100x 
-plan lens, filter sets for YFP and CFP fluorescence, and a cooled CCD Hamamatsu Orca camera. Openlab 3.0 software was used for image acquisition and processing.
Image analysis and measurements
Using MatLab software (MathWorks), we developed automated image analysis programs to facilitate localization of fluorescent foci in large numbers of cells as well as for analysis of other types of fluorescence signals. Briefly, during data collection, sets of phase-contrast and fluorescence images were collected for each field of cells. Segmentation of the phase-contrast images into individual cells was done by binary thresholding followed by region detection and shape filters. For each cell body, morphological shrinking operations were performed on the binary image to create a central skeleton that perfectly represented the individual curvature of the cell. The poles were calculated as the two points on each side of the long axis of the cell that were most distant from the midpoint of the skeleton (similar to an approach for identifying poles in Viollier et al. 2004). The gap between the poles and the skeleton was filled in by linear interpolation to create a complete curved midline. For each cell body, the corresponding region from the fluorescence image was analyzed and foci were detected by determining points of local maxima in fluorescence intensity. The position of each focus in the cell was determined by finding the closest point on the curved midline and measuring along the curve to each pole. In Figure 5C, the average ParAI-CFP intensity along the curved midline was calculated for each cell. For each pixel along the midline axis, the fluorescence intensity of all of the pixels in the cell lying along the perpendicular line from that point were summed and then averaged. This information is represented as an average intensity along the long axis of the cell and plotted against the position of YFP-ParBI foci in the same cell. Deconvolution of ParAI-CFP fluorescence was performed with Velocity software (Improvision) using a calculated point-spread function on an image-stack of 0.1-µm sections.
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E-MAIL matthew.waldor{at}tufts.edu; FAX (617) 636-2723. 
Supplemental material is available at http://www.genesdev.org.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1496506
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