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1 JB Accepts, published online ahead of print on 21 November 2008 J. Bacteriol. doi: /jb Copyright 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Growth conditions regulate the requirements for Caulobacter chromosome segregation Conrad W. Shebelut 1, Rasmus B. Jensen 2, and Zemer Gitai 1 Denmark. 1 Department of Molecular Biology, Princeton University, Washington Rd, Princeton, NJ Department of Science, Systems and Models, Roskilde University, Universitetsvej 1, DK-4000 Roskilde, Denmark. Present address: LEO Pharma, Industriparken 55, DK-2750 Ballerup, 1

2 Abstract Growth environments are important metabolic and developmental regulators. Here we demonstrate a growth environment-dependent effect on Caulobacter chromosome segregation of a small molecule inhibitor of the MreB bacterial actin cytoskeleton. Our results also implicate ParAB as important segregation determinants, suggesting that multiple distinct mechanisms can mediate Caulobacter chromosome segregation and that their relative contributions can be environmentally regulated. 2

3 One of the most fundamental biological processes is the faithful replication and segregation of genetic material during cell division. The MreB actin homolog and the ParAB system have emerged as two candidates that could mediate bacterial chromosome segregation (19). However, different studies have reported differences in the necessity for chromosome segregation of both MreB and ParAB in various model systems. For example, early studies that MreB plays an important role in Escherichia coli, Bacillus subtilis, Caulobacter crescentus, and Vibrio cholerae chromosome segregation (9, 13, 14, 17, 18) have been recently contradicted by reports that MreB is not essential for either E. coli or B. subtilis chromosome segregation (2, 7, 12). Similarly, while ParA and ParB plasmidic homologs have been clearly shown to mediate plasmid segregation (1, 4), the specific functions of chromosomally-encoded ParAB remain unclear and studies from different species report differences in the extent of their roles in chromosome positioning and/or translocation (3, 5, 6, 10, 16, 21, 23). Here we address a potential origin of these differences by examining the influence of growth conditions on chromosome segregation in Caulobacter crescentus and by using a rapid and specific chemical antagonist of MreB, A22 (S-(3,4-Dichlorobenzyl)isothiourea). A22 was originally identified as a compound that increased the frequency of anucleate cells in E. coli (11), and was subsequently found to specifically perturb MreB in Caulobacter, E. coli, and V. cholerae (9, 13, 18). Caulobacter represents an ideal model system for studying bacterial chromosome segregation since replication occurs once and only once per cell cycle and the ready ability to isolate swarmer cells that contain a single chromosome enables chromosome segregation to be followed in synchronized cell populations. A22 has different effects on chromosome dynamics when cells are grown in liquid or on solid media. Before replication initiates in the Caulobacter cell cycle, the single origin of 3

4 replication (ori) is localized to a cell pole. Soon after ori replication, one of the two oris is rapidly translocated to the opposite cell pole, such that ori localization provides a powerful assay for Caulobacter chromosome segregation. We followed ori localization using the strain MT174 (20), which contains as its only copy of ParB a functional GFP-ParB fusion that rescues the parb viability and division defects. ParB specifically binds to ori-proximal DNA sequences, such that this strain serves as a faithful reporter for ori localization (16, 20). To examine the effect of growth conditions on Caulobacter chromosome positioning, GFP-ParB cells were synchronized by density centrifugation and then grown in either liquid media or on 1% solid agarose pads in the presence of either 10 µg/ml A22 or the equivalent volume of the methanol diluent. This concentration of A22 is above the Minimum Inhibitory Concentration (MIC) for A22 for growth in both liquid and solid media (see below for details) and is sufficient to delocalize MreB (9). The percent of cells with bipolar GFP-ParB foci was scored at regular intervals in each of these four growth conditions. Since imaging the presence and location of GFP-ParB foci must be performed on solid pads, the cells grown in liquid were transferred to pads and rapidly imaged. In these experiments the cells exposure to the pad (2-3 minutes) was significantly shorter than the time required for completing ori translocation (~6-8 minutes (22)). Cells grown on agarose pads rapidly translocated the newly-replicated ori without A22 treatment, but showed a severe block in ori translocation in the presence of A22 (Fig 1a,c). Some cells also exhibited oris that did move away from the initial pole but failed to find the opposite pole, consistent with a defect in ori targeting (Fig 1c). Surprisingly, however, A22- treatment of cells grown in liquid media resulted in a delay, but not a block of ori translocation (Fig 1b,c). The above experiments were repeated with a strain, LS3827, harboring a laco array near the ori. Induced LacI-CFP was used to visualize the laco-labeled ori. This strain showed 4

5 ori dynamics similar to MT174 in liquid and solid growth conditions, both with and without A22 treatment (Fig S1). These results demonstrate that A22 blocks origin dynamics of cells grown in solid conditions, but only mildly delays origin dynamics of cells grown in liquid media. Differences in A22-dependant chromosome dynamics are not due to differential MreB perturbations. To examine whether the differential effects of A22 on chromosome positioning during solid and liquid growth are due to differential effects of A22 on MreB, we determined whether other A22-induced phenotypes such as growth, cell shape, and MreB delocalization are also more severe during solid as opposed to liquid growth. The growth of Caulobacter cells was more sensitive to A22 when cells were grown in liquid media than on solid media, with MICs of 1.25 µg/ml and 2.5 µg/ml respectively. Since GFP-MreB is only partially delocalized by 2.5 µg/ml of A22 but completely delocalized by 10 µg/ml of A22 (data not shown), we have used 10 µg/ml of A22 for the remainder of our experiments. Similar to the MIC results, cells grown overnight in the presence of A22 in liquid and solid conditions exhibited more pronounced cell shape defects in liquid media (Fig 2c & 2d). Finally, as detailed in the supplementary materials, we found that A22 had a similar effect on the localization of a GFP-MreB fusion in both liquid and solid growth. Thus, of all A22-induced phenotypes, chromosome positioning is the only one that is specifically exacerbated by growth on solid media, and most other phenotypes are more pronounced during liquid growth. Consequently, the differences in A22 s effect on ori positioning in the two growth conditions are not due to differential effects on MreB, but rather reflect differential requirements for ori translocation. In addition, the inverse correlations of A22 s effects on cell shape and chromosome positioning indicate that the chromosome positioning defects observed during growth on solid pads are not a secondary consequence of cell shape perturbations. 5

6 A22 treatment and ParB depletion have a synthetic effect on chromosome segregation in liquid. The presence of two different mechanisms for chromosome segregation could explain the lack of effect of A22 treatment observed under liquid culture conditions. One likely candidate for such a second mechanism is the Par system, which has been implicated in plasmid and chromosome dynamics in a number of species (10, 16, 23). In Caulobacter, the ParA and ParB proteins are essential due to their role in cell division (20). Overexpression of ParA or ParB results in a mild segregation defect (16), while overexpression of a dominantnegative ParA point mutant strongly inhibits the completion of chromosome segregation (21). To study ori dynamics upon Par system perturbation, we used a strain, MT148, that allows for conditional xylose-dependent expression of parb while imaging ori localization with a YFP fusion to another protein that colocalizes with the ori, MipZ (20). In order to study the role of the Par system in chromosome segregation, cells were depleted of ParB for 5 hours before the experiment, which resulted in ~3-fold reduction in ParB levels (Fig S2) (15, 20). Consistent with our previous results, MT148 cells grown in liquid without ParB depletion rapidly translocated their origins in the absence of A22, and A22 treatment delayed but did not block ori translocation (Fig 3a,b). Cells depleted of ParB in the absence of A22 showed a delay in origin translocation similar to that of A22 treatment alone (Fig 3a,b). This delayed ori translocation could reflect either the incomplete depletion of ParB in these cells or the presence of a second segregation mechanism that can partially compensate for the reduced ParB. When ParB depletion was combined with A22 treatment, cells showed a dramatically more severe translocation defect than either ParB depletion or A22 treatment alone (Fig 3a,b). This synthetic interaction between ParB depletion and A22 treatment raises the 6

7 possibility that these two perturbations target different aspects of the process of chromosome segregation. The combined effect of A22 treatment and ParB depletion is on ori translocation and not replication. The previous experiment demonstrates that the combination of A22 treatment and ParB depletion causes a severe defect in Caulobacter ori dynamics. However, our assay follows origins in situ, such that our observation that these cells generally retain a single ori focus cannot resolve a defect in chromosome segregation from a defect in chromosome replication. To distinguish these possibilities, we developed a method for using microscopy and flow cytometry to assay ori localization and DNA content respectively in the same cell population (see supplementary materials for details). The microscopy and flow profile for ParBdepleted cells treated with A22 for 3 hours after synchronization demonstrated that while these cells primarily contained single polar MipZ-YFP foci, they were overwhelmingly in the fully replicated (2N) state (Fig S3). These results suggest that there is no block in chromosomal replication as a result of combined depletion of ParB and A22 treatment, and that the synthetic defect in ori dynamics results from a defect in ori segregation. The ability to examine both the DNA content and ori positioning of the same cell population also enabled us to determine whether the A22-induced delay in the ori positioning of cells grown in liquid media (Fig 1) reflects delayed replication or segregation. We found that these cells exhibited a delay in the initiation of chromosome replication which correlated with their delayed ori positioning (Fig S4). Since flow cytometry must be performed in liquid, we have not been able to directly assess DNA replication in cells grown on solid pads. Together, our data suggest that when cells are grown in liquid media, A22 treatment can disrupt ori 7

8 translocation without blocking DNA replication, but only when ParB is also simultaneously perturbed. Does MreB function in chromosome segregation? The potential role of MreB in mediating bacterial chromosome segregation has recently emerged as a controversial topic. Early reports suggested that MreB plays an important role in segregating the chromosomes of E. coli, B. subtilis, Caulobacter, and V. cholerae (9, 13, 14, 17, 18). However, more recent studies in both E. coli and B. subtilis have failed to support the role of MreB in chromosome segregation (2, 7, 12). Our discovery that A22 treatment alone does not significantly affect Caulobacter ori translocation when cells are grown in liquid medium is consistent with the latter reports that MreB is not absolutely necessary for segregation in other species. At the same time, our results that the combination of A22 treatment and ParB depletion strongly perturbs ori translocation without blocking replication raise the possibility that the existence of multiple segregation mechanisms mask the role of MreB in mediating segregation in different contexts. This hypothesis is further supported by the fact that mreb appears to be the specific target of A22 in Caulobacter and other bacterial species (9, 13, 18), and that partially A22-resistant mreb point mutants shift the dose-response curve for A22 s effect on ori translocation (data not shown). However, it is still formally possible that the A22 effects we have observed are a consequence of the influence of A22 on another cellular target and that MreB does not play a significant role in Caulobacter chromosome dynamics. Multiple pathways can mediate chromosome segregation. The combined effects of A22 treatment and ParB depletion on the ori dynamics of Caulobacter cells grown in liquid media suggest that multiple pathways can collaborate in a partially redundant fashion to mediate chromosome segregation. In light of the essential nature of chromosome segregation, it is 8

9 perhaps not surprising that multiple mechanisms have evolved as safeguards. Nevertheless, it is surprising that this partial redundancy is only observed under specific growth conditions. It is possible that under stressed conditions neither pathway is sufficient, rendering each pathway necessary. Since different methods for visualizing chromosomal loci in vivo with different DNA-binding proteins might put different amounts of strain on the system, this scenario could reconcile conflicting reports about the effects of A22 on E. coli chromosome segregation (such as (13) and (12)). The relevant difference between liquid and solid growth conditions remains unclear. Preliminary experiments suggest that the impact of A22 on chromosome segregation is not affected by differences in oxygen levels or viscosity. In the future, it will prove interesting to determine what physiological parameters regulate chromosome segregation, how they are sensed, and how they exert their effect. Detailed materials and methods for the experiments described here can be found in the supplementary materials. We would like to acknowledge the members of the Gitai lab, Tom Silhavy, and Coleen Murphy for their helpful suggestions, Martin Thanbichler and James Gober for materials, and Christina DeCoste for assistance with the flow cytometry. This work was in part supported by funds from the U.S. Department of Energy Office of Science (BER, grant no. DE-FG02-05ER64136). RBJ thanks the Danish Natural Science Research Council for funding. 9

10 Figure Legends Fig 1. Effect of A22 treatment on ori segregation under solid and liquid growth conditions (a + b) Percent of cells (MT174) with bipolar GFP-ParB foci at different times after synchronization. Cells were grown with (red) and without (blue) 10 µg/ml A22 on M2G agarose pads (a) or in liquid M2G medium (b). Error bars indicate standard deviation (n > 100). (c) Representative images of the overlay of phase and GFP micrographs are shown for fields from the 0 minute (left), 60 minute (center), and 120 minute (right) timepoints from the experiments in (a) and (b). The arrow points to a cell with a defect in ori translocation. Scale bar represents 1 µm. Fig 2. Effects of A22 on Caulobacter morphology and MreB localization in liquid vs. solid media A strain expressing GFP-MreB was exposed to 10 µg/ml A22 in liquid M2G medium and on M2G agarose pads. Phase (top) and GFP (bottom) images were taken of cells grown in liquid medium (a + c) or on pads (b + d) with (c + d) or without (a + b) A22 treatment for 3 minutes, 90 minutes or overnight (O/N). Scale bar represents 1 µm. Fig 3. Synthetic effects of A22 treatment and ParB depletion on ori segregation (a) Percent of cells (MT148) with bipolar MipZ-YFP foci at different times after synchrony for cells untreated (blue), A22-treated (black), 5 hours ParB-depleted (red), or both A22-treated and ParB-depleted (green). Error bars indicate standard deviation (n > 100). (b) Representative images of the overlay of phase and YFP micrographs are shown for fields from the 0 minute (left) and 45 minute (right) timepoints from the experiment in (a). Scale bar represents 1 µm. 10

11 TABLE 1. C. crescentus strains Strain Relevant characteristic(s) Reference MT174 egfp-parb (20) MT148 pxyl-parb, eyfp-mipz (20) LS3814 pxyl-gfp-mreb (8) LS3827(MO1) (LacO) n ::CC0006; pxyl-laci-cfp, tetr-yfp (22) 11

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13 17. Soufo, H. J., and P. L. Graumann Actin-like proteins MreB and Mbl from Bacillus subtilis are required for bipolar positioning of replication origins. Curr Biol 13: Srivastava, P., G. Demarre, T. S. Karpova, J. McNally, and D. K. Chattoraj Changes in nucleoid morphology and origin localization upon inhibition or alteration of the actin homolog, MreB, of Vibrio cholerae. J Bacteriol 189: Thanbichler, M., and L. Shapiro Getting organized--how bacterial cells move proteins and DNA. Nat Rev Microbiol 6: Thanbichler, M., and L. Shapiro MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter. Cell 126: Toro, E., S. H. Hong, H. H. McAdams, and L. Shapiro Caulobacter requires a dedicated mechanism to initiate chromosome segregation. Proc Natl Acad Sci U S A 105: Viollier, P. H., M. Thanbichler, P. T. McGrath, L. West, M. Meewan, H. H. McAdams, and L. Shapiro Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication. Proc Natl Acad Sci U S A 101: Wu, L. J., and J. Errington RacA and the Soj-Spo0J system combine to effect polar chromosome segregation in sporulating Bacillus subtilis. Mol Microbiol 49:

14 Percent of Cells With Bipolar Foci a) b) 120% 100% 80% 60% 40% 20% 0% 120% 100% 80% 60% 40% c) Pads Liquid NO A22 A22 NO A22 A22 0 min 60 min 120 min -A22 +A22 -A22 PADS LIQUID 20% 0% A22 Time (Minutes)

15 -A22 +A22 Time After Addition of A22 (10ug/ml) 3 min 90 min O/N 3 min 90 min O/N a c d b Growth in Liquid Growth on Pads

16 a) b) Percent of Cells with Bipolar Foci -A22 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% M2G Liquid No Depletion, -A22 No Depletion, +A22 ParB Depleted, -A22 ParB Depleted, +A Time (Minutes) 0 min 45 min 0 min 45 min +A22 No Depletion Depletion