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Replication Initiator Dnaa Binds at the Caulobacter Centromere and Enables Chromosome Segregation

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Replication Initiator Dnaa Binds at the Caulobacter Centromere and Enables Chromosome Segregation Replication initiator DnaA binds at the Caulobacter centromere and enables chromosome segregation Paola E. Mera, Virginia S. Kalogeraki, and Lucy Shapiro1 Department of Developmental Biology, Beckman Center, Stanford University School of Medicine, Palo Alto, CA 94305 Contributed by Lucy Shapiro, October 1, 2014 (sent for review September 5, 2014; reviewed by James W. Gober and Matthew K. Waldor) During cell division, multiple processes are highly coordinated to number of ori (Fig. 1A). In a wild-type background, initiation of faithfully generate genetically equivalent daughter cells. In bacte- DNA replication results in the migration of one copy of the CFP- ria, the mechanisms that underlie the coordination of chromosome ParB/parS complex toward the opposite end of the cell, culmi- replication and segregation are poorly understood. Here, we nating in the establishment of a second fluorescent ParB focus at report that the conserved replication initiator, DnaA, can mediate the distal pole (7, 11) (Fig. 1 A and B). To investigate the de- chromosome segregation independent of replication initiation. It pendency of chromosome segregation on DNA replication, we does so by binding directly to the parS centromere region of the imaged the translocation of CFP-ParB/parS foci in cells expressing chromosome, and mutations that alter this interaction result in subphysiological DnaA concentrations. We constructed a Caulobacter cells that display aberrant centromere translocation and cell divi- strain in which transcription of the sole copy of dnaA was controlled sion. We propose that DnaA serves to coordinate bacterial DNA by the tightly regulated, xylose-inducible PxylX promoter (15) and in parB replication with the onset of chromosome segregation. which was replaced with a translational fusion of the gene encoding CFP to parB. When xylose was present in the growth media in this dnaA depletion strain, DnaA was expressed and DnaA | chromosome segregation | replication | Caulobacter | centromere fluorescent imaging revealed CFP-ParB localized to both cell poles, indicating replication initiation had occurred (Fig. 1C). ell division requires the faithful transmission of genetic in- When these cells were shifted to media lacking xylose, a single Cformation to each daughter cell. Thus, in all forms of life, CFP-ParB focus remained at the stalked pole (Fig. 1D), consis- multiple mechanisms cooperate to ensure that DNA synthesis tent with the requirement of DnaA to initiate replication (16). MICROBIOLOGY and chromosome segregation are temporally controlled and co- To characterize the effect of limited DnaA levels on chro- ordinated. Unlike eukaryotes, in which chromosomes are fully mosome segregation, we constructed a dnaA depletion strain replicated and organized into higher order structures before in which dnaA expression was driven by the leaky, vanillate- segregation (1), most bacteria segregate their chromosomes inducible PvanA promoter. Growth of this strain in the absence of progressively during replication (2). DnaA is a conserved bac- vanillate led to an 80% reduction in relative levels of DnaA after terial protein responsible for the initiation of DNA synthesis at 1 h, whereas DnaA levels decreased below the detection thresh- dnaA the chromosomal origin of replication (ori) (3, 4). The mecha- old in a PxylX- strain within 30 min following removal of inducer (Fig. 1G and Fig. S1). In the presence of vanillate nism by which chromosome segregation is initiated in bacteria is (250 μM), the dynamic localization of CFP-ParB during the cell less well understood. cycle was indistinguishable from that observed in a wild-type Although the factors responsible for DNA replication are highly strain (Fig. 1E). Growth in the absence of vanillate produced conserved among bacterial species, multiple mechanisms have cells with a single CFP-ParB focus. Notably, in this background, been proposed to account for chromosome segregation (5). In the we found that this single focus translocated to the opposite pole G1 phase of the Caulobacter crescentus cell cycle, the centromeric in more than 60% of the cells (Fig. 1F). region of the chromosome (parS) is tethered to one pole of the cell (Fig. 1A). Upon the swarmer to stalked cell transition, repli- cation initiates with replisome assembly at the origin of replica- Significance tion. The Par system in Caulobacter includes parS and two partitioning proteins, ParA and ParB. The ParB protein binds to DnaA is an essential and conserved bacterial protein that enables parS (6, 7), which, in turn, interacts with the nucleoid-associated the initiation of DNA replication. Although it is commonly held ParA ATPase to effect centromere movement (8–11). that the onset of bacterial chromosome segregation depends In Vibrio cholerae and Bacillus subtilis, the chromosome parti- on the initiation of DNA replication, we have found that in tioning protein ParA (Soj) has been reported to regulate replica- Caulobacter crescentus, chromosome segregation can be in- tion initiation by directly interacting with the DnaA replication duced in a DnaA-dependent, yet replication-independent manner. initiator protein, suggesting a connection between segregation and The chromosome replication origin, containing essential DnaA the initiation of replication (12–14). However, the signals that binding motifs, resides 8 kb from the centromere parS region that trigger the Par system to initiate chromosome segregation are not also contains DnaA binding motifs. The centromere parS region known. By generating Caulobacter strains that express limited bound to the ParB partition protein initiates movement across the concentrations of DnaA, we sought to determine whether repli- cell followed by the origin region. Mutations in a centromere cation initiation is a prerequisite for the translocation of the cen- DnaA motif that alter DnaA–centromere interaction exhibit aber- tromere complex. Under these conditions, we were able to detect rant patterns of ParB/parS translocation, implicating DnaA in the translocation of the chromosome in the absence of replication. We process of chromosome segregation. show that DnaA binds directly within the parS region and that altering binding of DnaA to parS leads to compromised chromo- Author contributions: P.E.M., V.S.K., and L.S. designed research; P.E.M. and V.S.K. per- some segregation. These results suggest that, in Caulobacter,DnaA formed research; P.E.M., V.S.K., and L.S. analyzed data; and P.E.M., V.S.K., and L.S. wrote plays a direct role in the initiation of chromosome segregation. the paper. Reviewers: J.W.G., University of California, Los Angeles; and M.K.W., Harvard Medical Results School, Brigham and Women’s Hospital. DnaA-Dependent Chromosome Translocation. In Caulobacter, the The authors declare no conflict of interest. ParB partitioning protein directly occupies the parS site, found 1To whom correspondence should be addressed. Email: [email protected]. 8 kb from ori (7). In cells expressing a functional CFP-ParB fu- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sion protein, the number of fluorescent foci reflects the copy 1073/pnas.1418989111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1418989111 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 A swarmer stalk ter ParB replisome 8 kb replication ParA-driven simultaneous parS G1 phase ori initiation segregation of parS replication & segregation B ParB-CFP 100 s Fig. 1. DnaA-dependent chromosome translocation. (A)DynamicsofCaulobacter chromosome segrega- M2G – % cell tion. (B F) Time-lapse fluorescence micrographs of wild type 0 h 0.5 h 1 h 1.5 h 2 h 2.5 h 3 h 0 1h 3h CFP-ParB in synchronized cells grown on M2G agarose C 100 pads (Left). Plots in Right depict fraction of synchro- nized cells with CFP-ParB translocated to the new pole s (reported as % cells) grown in liquid media. Samples were taken every 30 min and imaged over a 3-h time- %cell M2G + xylose ± 0 1h 3h course. Data are represented as mean SD from two independent experiments with n equals on average 100 D 200 cells per time point per experiment. Caulobacter s cells have a faster generation time in liquid media ell c than on agarose pads. (B) Wild-type cells (parB::cfp- M2G dnaA:: xylX::dnaA parB; MT190). Cells with dnaA expression regulated 0 1h 3h by PxylX (parB::cfp-parB, dnaA::Ω, xylX::dnaA; LS5368) + E 100 grown on M2G 0.3% xylose (C) or M2G only (D). Cells with dnaA expression regulated by PvanA (parB:: s cfp-parB, dnaA::Ω,vanA:dnaA;LS5369) grown on ell M2G + 250 μM vanillate (E) or M2G only (F); arrows %c indicate segregation of CFP-ParB/parS.(G) Vanil- 0 1h 3h M2G + vanillate late promoter has leaky expression of DnaA. Rela- 100 F tive levels of DnaA were followed by using s% Western blots with anti-DnaA antibodies (1 in 10,000 dilution) in mixed population of LS101 (Top), M2G dnaA:: vanA::dnaA %cell LS5368 (Middle), and LS5369 (Bottom) undergoing 0 1h 3h DnaA depletion. Cells were washed three times and G DnaA levels over 3 h depletion H Translocation frequency of ParB focus grown on liquid M2G. Exposures of all samples were 60 done simultaneously. (H) DnaA-dependent centro- Depletion of DnaA mere translocation. Plotted are the percentages of before after nducer 40 cells (parB::cfp-parB, dnaA::Ω, xylX::dnaA; LS5368) wash wash 0.5h 1h 1.5h 2h 3h with a single CFP-ParB focus translocated to the dnaA 3h + i 20 distant pole as a function of levels of xylose inducer xyl % cells added (i.e., varying subphysiological levels of DnaA). van 0 2468 Localization of CFP-ParB was determined by promoter % xylose (10-3 mg/mL) fluorescence microscopy of cells grown in M2G liq- uid media with the respective xylose concentration. These observations raised the possibility that DnaA is com- Limited Levels of DnaA Are Sufficient To Initiate Chromosome petent to promote the initiation of chromosome segregation Segregation but Not Replication.
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