Skip to main content

Main menu

  • Home
  • Articles
    • Current
    • Special Feature Articles - Most Recent
    • Special Features
    • Colloquia
    • Collected Articles
    • PNAS Classics
    • List of Issues
  • Front Matter
    • Front Matter Portal
    • Journal Club
  • News
    • For the Press
    • This Week In PNAS
    • PNAS in the News
  • Podcasts
  • Authors
    • Information for Authors
    • Editorial and Journal Policies
    • Submission Procedures
    • Fees and Licenses
  • Submit
  • Submit
  • About
    • Editorial Board
    • PNAS Staff
    • FAQ
    • Accessibility Statement
    • Rights and Permissions
    • Site Map
  • Contact
  • Journal Club
  • Subscribe
    • Subscription Rates
    • Subscriptions FAQ
    • Open Access
    • Recommend PNAS to Your Librarian

User menu

  • Log in
  • My Cart

Search

  • Advanced search
Home
Home
  • Log in
  • My Cart

Advanced Search

  • Home
  • Articles
    • Current
    • Special Feature Articles - Most Recent
    • Special Features
    • Colloquia
    • Collected Articles
    • PNAS Classics
    • List of Issues
  • Front Matter
    • Front Matter Portal
    • Journal Club
  • News
    • For the Press
    • This Week In PNAS
    • PNAS in the News
  • Podcasts
  • Authors
    • Information for Authors
    • Editorial and Journal Policies
    • Submission Procedures
    • Fees and Licenses
  • Submit
Research Article

Bacillus subtilis chromosome organization oscillates between two distinct patterns

Xindan Wang, Paula Montero Llopis, and David Z. Rudner
  1. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115

See allHide authors and affiliations

PNAS September 2, 2014 111 (35) 12877-12882; first published July 28, 2014; https://doi.org/10.1073/pnas.1407461111
Xindan Wang
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Paula Montero Llopis
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
David Z. Rudner
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: rudner@hms.harvard.edu
  1. Edited by Sharon R. Long, Stanford University, Stanford, CA, and approved July 2, 2014 (received for review April 23, 2014)

  • Article
  • Figures & SI
  • Info & Metrics
  • PDF
Loading

Significance

In bacteria, faithful and efficient DNA segregation is intimately linked to the spatial organization of the chromosome. Two distinct organization patterns have been described for bacterial chromosomes (ori-ter and left-ori-right) that appear to arise from distinct segregation mechanisms. Here, we show that the Bacillus subtilis chromosome oscillates between them during a replication–segregation cycle. Our data further suggest that the highly conserved condensin complex and the parABS partitioning system function as the core components that underlie these alternating patterns. We propose that this oscillatory cycle enhances the efficiency of DNA replication and sister chromosome segregation and provides a unifying model for the diverse patterns of chromosome organization observed in bacteria.

Abstract

Bacterial chromosomes have been found to possess one of two distinct patterns of spatial organization. In the first, called “ori-ter” and exemplified by Caulobacter crescentus, the chromosome arms lie side-by-side, with the replication origin and terminus at opposite cell poles. In the second, observed in slow-growing Escherichia coli (“left-ori-right”), the two chromosome arms reside in separate cell halves, on either side of a centrally located origin. These two patterns, rotated 90° relative to each other, appear to result from different segregation mechanisms. Here, we show that the Bacillus subtilis chromosome alternates between them. For most of the cell cycle, newly replicated origins are maintained at opposite poles with chromosome arms adjacent to each other, in an ori-ter configuration. Shortly after replication initiation, the duplicated origins move as a unit to midcell and the two unreplicated arms resolve into opposite cell halves, generating a left-ori-right pattern. The origins are then actively segregated toward opposite poles, resetting the cycle. Our data suggest that the condensin complex and the parABS partitioning system are the principal driving forces underlying this oscillatory cycle. We propose that the distinct organization patterns observed for bacterial chromosomes reflect a common organization–segregation mechanism, and that simple modifications to it underlie the unique patterns observed in different species.

  • DNA replication
  • ParA
  • chromosome segregation
  • SMC condensin

Central to reproduction is the faithful segregation of replicated chromosomes to daughter cells. In eukaryotes, DNA replication, chromosome condensation, and sister chromatid segregation are separated into distinct steps in the cell cycle that are safeguarded by checkpoint pathways. In bacteria, these processes occur concurrently, posing unique challenges to genome integrity and inheritance (1, 2). In the absence of temporal control, bacteria take advantage of spatial organization to promote faithful and efficient chromosome segregation. The organization of the chromosome dictates where the chromosome is replicated, and the factors that organize and compact the newly replicated DNA play a central role in its segregation (1, 2).

Studies in different bacteria have revealed strikingly distinct patterns of chromosome organization that appear to arise from different segregation mechanisms. In Caulobacter crescentus and Vibrio cholerae chromosome I, the origin and terminus are located at opposite cell poles, with the two replication arms between them, in a pattern referred to as “ori-ter” (3⇓–5). After replication initiation, one of the sister origins is held in place and the other is actively translocated to the opposite cell pole, regenerating the ori-ter organization in both daughter cells (5⇓⇓⇓⇓⇓–11). By contrast, in slow-growing Escherichia coli, the origin is located in the middle of the nucleoid and the two replication arms reside in opposite cell halves, in a “left-ori-right” pattern (12, 13). Replication initiates at midcell, and the duplicated origins segregate to the quarter positions followed by the left and right arms on either side, regenerating the left-ori-right pattern in the two daughter cells.

Although chromosome organization was first analyzed in the Gram-positive bacterium Bacillus subtilis, our understanding of the replication–segregation cycle in this bacterium has remained elusive. In pioneering studies, it was shown that during spore formation, the replicated origins reside at opposite cell poles and the termini at midcell in an ori-ter ter-ori organization (14⇓⇓⇓–18). A similar ori-ter pattern was observed during vegetative growth (15, 19). However, in separate studies, DNA replication was found to initiate at a midcell-localized origin (20, 21). How these disparate patterns fit into a coherent replication–segregation cycle has never been addressed and motivated this study. Our analysis has revealed that the B. subtilis chromosome follows an unexpected and previously unidentified choreography during vegetative growth in which the organization alternates between ori-ter and left-ori-right patterns. Our data further suggest that the highly conserved partitioning system (parABS) and the structural maintenance of chromosomes (SMC) condensin complex, in conjunction with replication initiation, function as the core components for this oscillating cycle. We propose that this cycle enhances the efficiency of DNA replication and sister chromosome segregation and provides a unifying model for the diverse patterns of chromosome organization observed in bacteria.

Results

Sporulating B. subtilis Chromosomes Have an ori-ter Organization.

As a first step toward our analysis of chromosome organization in B. subtilis, we modified the cytological tools to visualize chromosomal loci in live cells. Previous fluorescently tagged repressor proteins bound to operator arrays caused partial or complete blocks to replisome progression in B. subtilis (15, 22, 23). We reduced the number of binding sites in the arrays and lowered expression of the repressor protein fusions using a weak constitutive promoter (Materials and Methods). Under these conditions, virtually all cells contained fluorescent foci with no detectable impact on growth rate, nucleoid morphology, or nucleoid size distribution (Fig. S1).

To validate these tools, we analyzed the organization of the replicated chromosomes during spore formation. At early stages in this developmental process, the replicated chromosomes adopt a structure called the axial filament, in which the origins are anchored at the cell poles and the termini are present near midcell (16, 24⇓–26). We visualized six chromosomal loci [−7° (ori) and ±87°, ±120°, and +174° (ter)] during sporulation and determined their population-average, normalized position relative to cell length using automated image analysis software (27) (Fig. 1 A–E). The origins were present close to the cell poles at 0.17 and 0.83, and the termini were located at midcell (0.5). The ±87° and ±120° loci localized linearly between them. These results confirm and extend previous observations (24, 25, 28) that the sporulating chromosomes are organized linearly along their length from the poles to midcell in a C. crescentus-like ori-ter ter-ori organization (Fig. 1B).

Fig. 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 1.

Chromosome organization during sporulation. (A–E) Exponentially growing cells were induced to sporulate and imaged after 2 h. All strains contained a mutation in spoIIIE (spoIIIE36) (55) to prevent DNA transport. (A) Chromosome map showing positions of operator arrays. (B) Schematic summary of cellular localization of loci. Representative micrographs of cells labeled with pairs of loci ori-ter (C, Left), ±87° (D, Left), and ±120° (E, Left) are shown. (C–E, Right) Loci position relative to cell length analyzed (Materials and Methods). The centroid of the Gaussian distribution is shown on the top of each histogram. (F and G) Origin number and localization in rich and poor growth media. Origins (green, tetO48/TetR-CFP), nucleoid (blue, HBsu-mYpet), and membrane [red, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl), pyridinium dibromide (FM4-64)] are shown. (Scale bar: 4 μm.)

B. subtilis Maintains a Partially Diploid State.

The analysis of chromosome organization in C. crescentus and slow-growing E. coli was facilitated by their simple cell cycles with no overlapping rounds of replication (3, 12, 13). When grown in rich media, B. subtilis, like E. coli, undergoes multifork replication and the cells are born with partially replicated chromosomes. In defined rich medium [doubling time (τ) = 35 min], the cells were born with two origins and, on average, the number of origins per cell was 3.1 (n = 1,825) (Fig. 1F). To obtain a simpler DNA replication cycle, we grew B. subtilis in minimal medium supplemented with different carbon sources, as was done previously in E. coli, to reduce growth rate and DNA content. Although we could slow growth in glucose (τ = 48 min), succinate (τ = 77 min), and sorbitol (τ = 98 min), under all conditions tested, the cells were born with two origins, and the average number of origins per cell was 2.8, 2.3, and 2.1 (n > 1,700), respectively (Fig. 1G and Fig. S2A). Even under the slowest growth condition, cells were born with greater than 50% of their chromosome replicated (Fig. S2B). Thus, we were unable to identify conditions in which cells are born with a single unreplicated chromosome (1-N content). These results suggest that there is a link between DNA replication and cell division that ensures cells are born with two almost completely replicated chromosomes under normal growth conditions, a requirement for successful sporulation. We therefore initiated our analysis using conditions in which cells were artificially restricted to a single copy of the chromosome by inhibiting replication initiation.

Left-ori-Right Chromosome Organization in Cells with a Single Chromosome.

To analyze vegetatively growing cells with 1-N DNA content, we inhibited replication initiation using a temperature-sensitive mutant of the helicase loader (DnaB) (29, 30). Cells were grown in rich medium at the permissive temperature (30 °C, τ = 57 min) to early exponential phase and then shifted to the restrictive temperature (42 °C, τ = 33 min) (Fig. 2A). After 1 h, 84% of the cells (n = 1,224) had a single-lobed nucleoid containing one replication origin, indicating that the chromosome content was reduced to 1-N (Fig. 2A). After 1.5 h, >90% of the cells (n = 2,399) had a single origin. Furthermore, as observed previously by McGinness and Wake (31), the nucleoids adopted a bilobed structure (Fig. 2A). Strikingly, in most cases (92%, n = 2,196), the origin was located at or close to the center of the two lobes (Fig. 2 A, C, D, and F). This localization pattern is dramatically different from sporulating cells, in which origins are at the extreme poles (Fig. 1C), and vegetatively growing cells with unperturbed replication, in which origins are frequently localized at the nucleoid periphery (14, 16) (Fig. 1 F and G and Fig. S2A). To determine whether each nucleoid lobe represents a chromosome arm, we visualized pairs of loci on the left and right arms. Eighty percent of the cells (n = 1,633) with fluorescently tagged loci at ±87° and 88% of the cells (n = 1,634) with tagged loci at ±120° had these markers in separate nucleoid lobes (Fig. 2 B and E). We also monitored loci at −87° and −120° on the same chromosome arm. In 86% of the cells (n = 1,804), the two loci were present in the same nucleoid lobe (Fig. 2 B and E). Furthermore, in the majority of these cells, the −87° locus was closer to the center of the two lobes and the −120° locus was closer to the nucleoid edge (Fig. 2 B and E).

Fig. 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 2.

Left-ori-right organization in cells with a single chromosome. Cells with a dnaB(ts) mutation were shifted from 30 °C to 42 °C for 1.5 h to block new rounds of initiation. (A) Origin localization before and after temperature shift. Origins (green), nucleoid (red), and membrane (white) are shown. (B) Representative micrographs of cells labeled at pairs of loci: ±87° (Left), ±120° (Center), and −87° and −120° (Right). (C) Cells labeled at ori (red) and ter (green). (D) Cells labeled at ori (red) and expressing RTP-YFP (green). (E and F) Quantitative analysis of loci positions. Origin localization in dnaB(ts) 1.5 h after temperature shift and degradation of SMC (G) or ScpB (H) is shown. (Scale bars: 4 μm.)

Analysis of a locus close to the replication terminus (+174°) revealed that most cells had a ter locus directly between the two lobes (64%, n = 2,196) or in close proximity to this position (17%) (Fig. 2 C and F). However, in 18% of the cells, the terminus was present near the edge of the nucleoid. To visualize a larger region of the terminus, we used YFP fusion to the replication termination protein (RTP) (32). RTP binds to nine termination sites spanning ∼430 kb of the terminus region (33). RTP-YFP localized as a single focus, clusters of foci, or multiple foci spanning the bilobed nucleoid (Fig. 2D), consistent with the terminus region connecting the ends of the two lobes. Similar results were obtained using different strain backgrounds and different growth media, as well as when initiation was blocked using dnaA(ts) or induction of SirA, an inhibitor of DnaA (34, 35). Collectively, these data indicate that under conditions in which B. subtilis is restricted to 1-N content, the nucleoid is organized in a pattern like the E. coli chromosome during slow growth, with the two arms present on either side of the origin and genetic loci arranged linearly from the middle toward the poles. Finally, the terminus region connects the two ends.

Origin-Localized SMC Complexes Are Required for Left-ori-Right Organization.

In E. coli, the left-ori-right organization is shifted to an ori-ter pattern in cells lacking the condensin complex MukBEF (36, 37). We used degradable variants of B. subtilis SMC or its partner protein ScpB to investigate whether the left-ori-right organization similarly depends on the condensin complex. Degradation of SMC (and ScpB separately) was induced when dnaB(ts) cells were shifted to the restrictive temperature to block replication initiation. After 1.5 h, 91% of SMC-degraded cells (n = 1,005) had a more extended chromosome structure, with the replication origin located at the extreme edge of the nucleoid (Fig. 2G and Fig. S3). Similar results were obtained after ScpB degradation (Fig. 2H). The Spo0J (ParB) protein in B. subtilis has been shown to recruit SMC complexes to the origin region (28, 38). Accordingly, we investigated the organization of the nucleoid in its absence (Fig. S3). In the majority of the Spo0J mutant cells (70%, n = 999), the origin of replication localized to the edge of the nucleoid. This phenotype was principally due to the absence of Spo0J-mediated recruitment of SMC rather than the loss of the parABS partitioning activity, because cells lacking Soj (ParA) that are not impaired in SMC recruitment (28) retained the left-ori-right pattern (Fig. S3). We conclude that origin-localized condensin complexes play a critical role in generating the E. coli-like pattern.

ori-ter and Left-ori-Right Patterns Alternate During the Replication–Segregation Cycle.

The two distinct patterns of chromosome organization described above were observed under extreme conditions. In one, the origins were physically tethered to the cell poles during bacterial differentiation (24, 26); in the other, replication initiation was blocked and the cells were restricted to 1-N content. To investigate whether exponentially growing cells with unperturbed DNA replication possess either pattern, we used time-lapse fluorescence microscopy to visualize chromosomal loci during replication–segregation cycles. To ensure the best time resolution, we used minimal medium supplemented with sorbitol (τ = 98 min). First, we visualized the origin (Fig. 3A). The cells were born with two origins at opposite edges of the nucleoid and remained at the nucleoid periphery for the rest of the replication cycle. Previous studies suggested that replication initiates in the middle of the nucleoid (15, 20). However, in our movies, the origin foci became brighter at or close to the nucleoid periphery, indicative of their replication at this site (Fig. 3A and Fig. S4A, red carets). Similarly, in snapshot images, replisomes colocalized with the brighter origin foci at the nucleoid edge (Fig. S4B). Shortly after their replication, the two new origins migrated as a unit to the middle of the nucleoid (Fig. 3A and Fig. S4A, yellow carets). This event was followed by their bidirectional segregation to opposite edges of the nucleoid. This cycle is consistent with snapshot images in which most cells have two origins at the outer edges of the nucleoid (16, 19) (Fig. 1 F and G and Fig. S2A).

Fig. 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 3.

Alternating patterns of chromosome organization during the cell cycle. (A) Time-lapse progression (5-min intervals) of cells labeled at the origin (green) and nucleoid (red) grown in minimal medium supplemented with sorbitol. Red carets highlight replication initiation at the edge of the nucleoid. Yellow carets show duplicated origins at the center of the nucleoid before their segregation. (B) Time-lapse progression (10-min intervals) of cells labeled at −87° (green) and +87° (red). Black arrows highlight resolution of the left and right loci. (C) Representative micrograph of cells labeled at three chromosomal loci: origin (blue, mCherry-Spo0J), −87° (green), and +87° (red). Stages in the replication–segregation cycle are indicated (I–III) and are interpreted in the schematic model (Right). Origin, −87°, and +87° loci are labeled as blue, green, and red balls, respectively. Chromosome arms are shown as gray lines. (Scale bar: 4 μm.)

Next, we tracked markers on the left and right chromosome arms. For most of the cell cycle, the ±87° loci localized adjacent to each other. However, for a brief period (20–30 min), the two loci separated, localizing on either side of the nucleoid (Fig. 3B, black arrow, and Fig. S5). After this period of resolution, the two loci were replicated, segregated, and colocalized again. Similar results were obtained for the pair of markers at ±120°. To relate the dynamic movement of the markers on opposite arms to the replication cycle, we used mCherry–Spo0J (ParB) fusion to localize the origin (14) while simultaneously following the markers at ±87°. When the ±87° markers colocalized, the origin was almost always present at the edge of the nucleoid (Fig. 3C). However, when the ±87° markers were spatially resolved, the replicated origins were either in the middle of the nucleoid between the ±87° markers or had already segregated to the edges of the nucleoid (Fig. 3C). Thus, movement of the duplicated origins to midcell is accompanied by resolution of the left and right arms. This unprecedented choreography can explain some of the unusual localization patterns observed previously (15, 19) and is fully supported by snapshot images in which we visualized fluorescently labeled replisomes and an origin locus (Fig. S6).

Combining the time-lapse microscopy, the three-color images, and the replisome localization relative to the origin, a picture emerges of the replication–segregation cycle in B. subtilis (Fig. 4). At birth, the two origins are located at opposite edges of the nucleoid, with the partially replicated arms lying side by side. At the time of the next round of initiation, replisomes assemble on the polarly localized origins. The newly replicated origins then migrate as a unit to the middle of the nucleoid. At this time, the unreplicated chromosome arms become spatially resolved and the replisomes track independently of each other along them (Fig. S7). The left-ori-right pattern of the template DNA persists even after the origins are segregated to the nucleoid periphery and only returns to the ori-ter–like organization after the arms themselves have been replicated.

Fig. 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 4.

Schematic model of the organization–segregation cycle in B. subtilis, and its comparison with C. crescentus and E. coli. Origins are represented as black balls, and termini are represented as brown lines in B. subtilis and E. coli and as a brown oval in C. crescentus. The compacted left and right chromosome arms are shown as thick blue and purple lines (or blobs). Newly replicated DNA is shown with a lighter hue, whereas uncompacted DNA is shown as thin lines. In the B. subtilis, newborn cell, unreplicated DNA is shown as a black cloud. The replication–segregation cycle of C. crescentus and E. coli corresponds to two halves of the cycle in B. subtilis.

Partitioning Locus Helps Establish and Maintain the Origins at the Nucleoid Edge.

We investigated whether the B. subtilis partitioning locus is responsible for maintaining the origins at the nucleoid periphery until the next round of replication. Chromosomally encoded par loci are composed of an ATPase (ParA), a DNA binding protein (ParB), and a centromere-like sequence (parS) (39). All three are critical for origin segregation in C. crescentus (40, 41), Pseudomonas aeruginosa (42), and V. cholerae chromosome II (43). In B. subtilis, cells lacking ParA, called Soj, have a very mild defect in chromosome segregation (23). ParB mutants have a more pronounced phenotype, but this is thought to be a consequence of a failure to enrich SMC complexes at the origin (23, 28, 38). Accordingly, for these experiments, we used a Soj (ParA) mutant that has no apparent impact on origin-localized SMC (28). Analysis of origin dynamics in cells lacking Soj revealed that the directional movement of the newly replicated origins was abolished (Fig. 5 A and C and Fig. S8). Instead of segregating to the edge of the nucleoid (Figs. 3A and 5B and Fig. S8), the origins moved more slowly and in an irregular manner (Fig. 5 A and C and Fig. S8). Although they reached the nucleoid edge, they frequently moved back toward midcell.

Fig. 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 5.

Impaired origin segregation and chromosome arm dynamics in the absence of Soj (ParA). (A) Time-lapse progression (5-min intervals) of ∆soj cells. The origin (green) and nucleoid (red) are shown. Representative origin traces in WT (B) and ∆soj (C) are shown. The position of the origin was plotted relative to the total cell length (Fig. S8). (D) Interfocal distances of ±87° loci are analyzed in representative WT (Upper) and ∆soj (Lower) cells in time-lapse progressions (10-min intervals). Each graph plots the distance between a pair of ±87° loci relative to the cell length (L) at indicated times during their replication cycle. For the purpose of this analysis, the replication cycle of these loci begins when both ±87° loci are replicated (defined as frame 1) and finishes when either of the two loci is replicated again. Because each cell has two pairs of ±87° loci in its replication cycle, the maximum distance of a pair of ±87° loci is 50% of the cell length (Fig. S10).

Next, we investigated whether Soj helps maintain the two chromosomal arms adjacent to each other in an ori-ter pattern. Time-lapse imaging of markers at ±87° in the ∆soj mutant revealed that these loci remain resolved for the majority (62 ± 8%) of the replication cycle (n = 100) (Fig. 5D and Figs. S9 and S10B). By contrast, in WT cells, these loci were resolved for only 37 ± 7% (n = 100) of the replication cycle (Figs. 3B and 5D and Figs. S5 and S10A). Furthermore, instead of alternating between arm colocalization and arm resolution as observed in WT cells (Figs. 3B and 5D and Figs. S5 and S10A), the ∆soj mutant had more dynamic arm movements, with irregular patterns of arm colocalization and resolution (Fig. 5D and Figs. S9 and S10B). These data indicate that Soj, and presumably the partitioning locus, facilitates bidirectional origin segregation and plays a central role in both establishing and maintaining the ori-ter pattern.

Discussion

Our data support a model in which origin-localized condensin complexes and the partitioning system are the principal driving forces that underlie the two patterns described here, whereas replication initiation serves as the switch that triggers the oscillation between them. In this model, recruitment of SMC complexes to the origin by ParB (28, 38) sets up the left-ori-right pattern. SMC resolves the replicated origins (44, 45), and by analogy to the stiffening of eukaryotic centromeres by condensin (46), we hypothesize that SMC rigidifies the origin region constraining the left and right arms on either side of the origin through lengthwise condensation (47). Because SMC complexes are present at the origin throughout the cell cycle, force must be applied to the chromosome to generate the ori-ter pattern. We have shown that Soj (ParA) actively segregates origins to the nucleoid periphery and plays an important role in both establishing and maintaining this ori-ter organization. By analogy to chromosome and plasmid segregation in other bacteria (48, 49), we hypothesize that Soj acts by exerting pulling forces on Spo0J (ParB) bound to parS adjacent to the origin. Finally, we propose that replication initiation triggers the change in organization pattern by transiently inactivating the forces exerted on the origins. One possibility is that Soj’s role in replication initiation (50) could temporarily relax its segregation function. Alternatively or in addition, replication of the origin region could inactivate the partitioning system by transiently disrupting the Spo0J–parS complexes. When force on the origins is lost, the chromosome resolves into its SMC-mediated left-ori-right pattern. Par-mediated segregation of the newly replicated origins initiates a new round of this oscillatory cycle. Intriguingly, in cells that are blocked for replication initiation, the partitioning system appears to become inactivated, leading to a stable left-ori-right configuration (Fig. 2). Chronic inhibition of replication initiation could maintain Soj in its replication-active state (50), resulting in a sustained loss of its segregation function. Alternatively, the loss of partitioning activity could be due to a change in the dynamic properties of the partitioning system (8, 11, 39, 48, 49) resulting from a reduced chromosome content in an enlarged cellular compartment. Systematic analysis of chromosome organization in P. aeruginosa (42) suggests that its replication–segregation cycle could follow an alternating pattern similar to the one described here. Interestingly, this bacterium contains both condensin complexes and the Par system, and it does not anchor its origins at the cell poles. Furthermore, recent quantitative analysis of snapshot images suggests an ori-ter–like chromosome organization in fast-growing E. coli (51). The localization of the replisome to midnucleoid zones in these fast-growing cells (51) raises the possibility that a transient left-ori-right intermediate might exist during these overlapping replication cycles. Time-lapse imaging of loci on both replication arms or simultaneous imaging of three loci will be required to establish whether or not this is the case.

In the cycle we report here, the template DNA adopts a left-ori-right organization, whereas the newly replicated DNA is segregated and organized in an ori-ter pattern. We hypothesize that this oscillating pattern enhances the efficiency of replication and segregation of the bacterial chromosome. The left-ori-right pattern ensures that the two replisomes track on the template DNA in opposite cell halves (Figs. S6 and S7), providing spatial resolution to the complex topologies that inevitably arise at the replication forks. The ori-ter pattern of the newly replicated DNA ensures that the sister chromosomes are segregated as far as possible from each other. Finally, in the specific case of B. subtilis, the partitioning system also ensures that the origins reside close to the poles, where they can be readily anchored if the cell enters the sporulation pathway (24, 26). Initiation of a new round of replication transiently blocks entry into sporulation (52), and our data suggest that it also releases the origins from their polar position, allowing the replication–segregation cycle to begin again.

Finally, our data raise the possibility that the disparate patterns of chromosome organization observed in bacteria arise from modifications to a common organization–segregation mechanism. In the case of E. coli, which lacks a partitioning locus, the condensin-mediated left-ori-right pattern is established shortly after origin segregation and this ground state is maintained throughout the replication–segregation cycle (Fig. 4). In the case of C. crescentus, V. cholerae chromosome I, and sporulating B. subtilis, distinct polar anchoring mechanisms “lock” chromosome organization in the ori-ter pattern, half of the full cycle (Fig. 4). Although the removal of the polar anchoring mechanism or addition of a par locus is unlikely to be sufficient to recover the second half of the respective cycles, we propose that these factors are the principal drivers in evolving distinct organization–segregation cycles.

Materials and Methods

General Methods.

B. subtilis strains were derived from the prototrophic strain PY79 . Cells were grown in defined rich casein hydrolysate medium (53) or minimal medium (S750) (54) supplemented with 1% glucose, sorbitol, or succinate as specified. To monitor chromosome organization during sporulation, we used a mutant of the SpoIIIE DNA translocase (spoIIIE36) that engages the forespore chromosome after polar division but is blocked in DNA transport (28, 55). Sporulation was induced by resuspension at 37 °C according to the method of Sterlini–Mandelstam (53). Images were taken 2 h after the initiation of sporulation. E. coli SspB protein was induced with 0.5% xylose for degradation of SsrA-tagged proteins (56). Strains, plasmids, and oligonucleotides used in this study are listed in Tables S1–S3. Strain and plasmid constructions are described in SI Materials and Methods.

Fluorescence Microscopy.

Fluorescence microscopy was performed with an Olympus BX61 microscope equipped with an UPLFLN 100×/1.3-N.A. phase-contrast oil objective and a CoolSnapHQ cooled CCD camera (Photometrics) or a Nikon Ti microscope equipped with Plan Apo 100×/1.4-N.A. phase-contrast oil objective and a CoolSnapHQ2 camera. Membranes were stained with trimethylammonium diphenylhexatriene (Molecular Probes) at 0.01 mM or with FM4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl), pyridinium dibromide; Molecular Probes] at 3 μg/mL. DNA was stained with DAPI (Molecular Probes) at 2 μg/mL. Images were cropped and adjusted using MetaMorph software (Molecular Devices). Final figure preparation was performed in Adobe Illustrator (Adobe Systems).

For snapshot imaging, cells were immobilized using 2% (wt/vol) agarose pads containing growth media. For time-lapse imaging, a glass-bottomed dish (Willco dish HBSt-5040; Willco Wells) was used as a coverslip. Exponentially growing cells were concentrated at 3,300 × g for 30 s. After removal of 90% of the supernatant, 2 μL of the culture was spotted onto the glass-bottomed dish. A 2% (wt/vol) agarose pad containing growth media was then laid on top of the bacteria. These cells were imaged on a Well Plate Holder stage (TI-SH-W; Nikon) equipped with a humid, temperature-controlled incubator (TC-MIS; Bioscience Tools). The upper face of the pad was fully exposed, allowing adequate oxygen for growth. The objective was heated using a Bioptechs objective heater system. Images were acquired every 5 or 10 min as specified. Image analyses were performed using the MathWorks MATLAB-based program MicrobeTracker (27). Details of image analysis can be found in SI Materials and Methods.

Acknowledgments

We thank members of the Bernhardt laboratory and the laboratory of D.Z.R. as well as Viknesh Sivanathan for stimulating discussions and support, and we thank Alan Grossman, Kevin Griffith, Dirk Landgraf, David Sherratt, and Rodrigo Reyes-Lamothe for plasmids. Support for this work comes from National Institutes of Health Grants GM086466 and GM073831 (to D.Z.R.). X.W. was a long-term fellow of the Human Frontier Science Program. P.M.L. is a Helen Hay Whitney postdoctoral fellow.

Footnotes

  • ↵1To whom correspondence should be addressed. Email: rudner{at}hms.harvard.edu.
  • Author contributions: X.W. and D.Z.R. designed research; X.W. performed research; X.W. and P.M.L. contributed new reagents/analytic tools; X.W. and D.Z.R. analyzed data; and X.W. and D.Z.R. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • See Commentary on page 12580.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1407461111/-/DCSupplemental.

References

  1. ↵
    1. Reyes-Lamothe R,
    2. Nicolas E,
    3. Sherratt DJ
    (2012) Chromosome replication and segregation in bacteria. Annu Rev Genet 46:121–143.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Wang X,
    2. Montero Llopis P,
    3. Rudner DZ
    (2013) Organization and segregation of bacterial chromosomes. Nat Rev Genet 14(3):191–203.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Viollier PH,
    2. et al.
    (2004) Rapid and sequential movement of individual chromosomal loci to specific subcellular locations during bacterial DNA replication. Proc Natl Acad Sci USA 101(25):9257–9262.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Fogel MA,
    2. Waldor MK
    (2005) Distinct segregation dynamics of the two Vibrio cholerae chromosomes. Mol Microbiol 55(1):125–136.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Fogel MA,
    2. Waldor MK
    (2006) A dynamic, mitotic-like mechanism for bacterial chromosome segregation. Genes Dev 20(23):3269–3282.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Bowman GR,
    2. et al.
    (2008) A polymeric protein anchors the chromosomal origin/ParB complex at a bacterial cell pole. Cell 134(6):945–955.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Ebersbach G,
    2. Briegel A,
    3. Jensen GJ,
    4. Jacobs-Wagner C
    (2008) A self-associating protein critical for chromosome attachment, division, and polar organization in caulobacter. Cell 134(6):956–968.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Schofield WB,
    2. Lim HC,
    3. Jacobs-Wagner C
    (2010) Cell cycle coordination and regulation of bacterial chromosome segregation dynamics by polarly localized proteins. EMBO J 29(18):3068–3081.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Shebelut CW,
    2. Guberman JM,
    3. van Teeffelen S,
    4. Yakhnina AA,
    5. Gitai Z
    (2010) Caulobacter chromosome segregation is an ordered multistep process. Proc Natl Acad Sci USA 107(32):14194–14198.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Yamaichi Y,
    2. et al.
    (2012) A multidomain hub anchors the chromosome segregation and chemotactic machinery to the bacterial pole. Genes Dev 26(20):2348–2360.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Ptacin JL,
    2. et al.
    (2010) A spindle-like apparatus guides bacterial chromosome segregation. Nat Cell Biol 12(8):791–798.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Nielsen HJ,
    2. Ottesen JR,
    3. Youngren B,
    4. Austin SJ,
    5. Hansen FG
    (2006) The Escherichia coli chromosome is organized with the left and right chromosome arms in separate cell halves. Mol Microbiol 62(2):331–338.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Wang X,
    2. Liu X,
    3. Possoz C,
    4. Sherratt DJ
    (2006) The two Escherichia coli chromosome arms locate to separate cell halves. Genes Dev 20(13):1727–1731.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Lin DC,
    2. Levin PA,
    3. Grossman AD
    (1997) Bipolar localization of a chromosome partition protein in Bacillus subtilis. Proc Natl Acad Sci USA 94(9):4721–4726.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Webb CD,
    2. et al.
    (1998) Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis. Mol Microbiol 28(5):883–892.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Webb CD,
    2. et al.
    (1997) Bipolar localization of the replication origin regions of chromosomes in vegetative and sporulating cells of B. subtilis. Cell 88(5):667–674.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Glaser P,
    2. et al.
    (1997) Dynamic, mitotic-like behavior of a bacterial protein required for accurate chromosome partitioning. Genes Dev 11(9):1160–1168.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Sharpe ME,
    2. Errington J
    (1998) A fixed distance for separation of newly replicated copies of oriC in Bacillus subtilis: Implications for co-ordination of chromosome segregation and cell division. Mol Microbiol 28(5):981–990.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Teleman AA,
    2. Graumann PL,
    3. Lin DC,
    4. Grossman AD,
    5. Losick R
    (1998) Chromosome arrangement within a bacterium. Curr Biol 8(20):1102–1109.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Lemon KP,
    2. Grossman AD
    (1998) Localization of bacterial DNA polymerase: Evidence for a factory model of replication. Science 282(5393):1516–1519.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Berkmen MB,
    2. Grossman AD
    (2006) Spatial and temporal organization of the Bacillus subtilis replication cycle. Mol Microbiol 62(1):57–71.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Bernard R,
    2. Marquis KA,
    3. Rudner DZ
    (2010) Nucleoid occlusion prevents cell division during replication fork arrest in Bacillus subtilis. Mol Microbiol 78(4):866–882.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Lee PS,
    2. Grossman AD
    (2006) The chromosome partitioning proteins Soj (ParA) and Spo0J (ParB) contribute to accurate chromosome partitioning, separation of replicated sister origins, and regulation of replication initiation in Bacillus subtilis. Mol Microbiol 60(4):853–869.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Wu LJ,
    2. Errington J
    (2003) RacA and the Soj-Spo0J system combine to effect polar chromosome segregation in sporulating Bacillus subtilis. Mol Microbiol 49(6):1463–1475.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Bogush M,
    2. Xenopoulos P,
    3. Piggot PJ
    (2007) Separation of chromosome termini during sporulation of Bacillus subtilis depends on SpoIIIE. J Bacteriol 189(9):3564–3572.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Ben-Yehuda S,
    2. Rudner DZ,
    3. Losick R
    (2003) RacA, a bacterial protein that anchors chromosomes to the cell poles. Science 299(5606):532–536.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Sliusarenko O,
    2. Heinritz J,
    3. Emonet T,
    4. Jacobs-Wagner C
    (2011) High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics. Mol Microbiol 80(3):612–627.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Sullivan NL,
    2. Marquis KA,
    3. Rudner DZ
    (2009) Recruitment of SMC by ParB-parS organizes the origin region and promotes efficient chromosome segregation. Cell 137(4):697–707.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Velten M,
    2. et al.
    (2003) A two-protein strategy for the functional loading of a cellular replicative DNA helicase. Mol Cell 11(4):1009–1020.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Rokop ME,
    2. Auchtung JM,
    3. Grossman AD
    (2004) Control of DNA replication initiation by recruitment of an essential initiation protein to the membrane of Bacillus subtilis. Mol Microbiol 52(6):1757–1767.
    OpenUrlCrossRefPubMed
  31. ↵
    1. McGinness T,
    2. Wake RG
    (1979) Completed Bacillus subtilis nucleoid as a doublet structure. J Bacteriol 140(2):730–733.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Lemon KP,
    2. Kurtser I,
    3. Grossman AD
    (2001) Effects of replication termination mutants on chromosome partitioning in Bacillus subtilis. Proc Natl Acad Sci USA 98(1):212–217.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Griffiths AA,
    2. Andersen PA,
    3. Wake RG
    (1998) Replication terminator protein-based replication fork-arrest systems in various Bacillus species. J Bacteriol 180(13):3360–3367.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Wagner JK,
    2. Marquis KA,
    3. Rudner DZ
    (2009) SirA enforces diploidy by inhibiting the replication initiator DnaA during spore formation in Bacillus subtilis. Mol Microbiol 73(5):963–974.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Rahn-Lee L,
    2. Gorbatyuk B,
    3. Skovgaard O,
    4. Losick R
    (2009) The conserved sporulation protein YneE inhibits DNA replication in Bacillus subtilis. J Bacteriol 191(11):3736–3739.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Badrinarayanan A,
    2. Lesterlin C,
    3. Reyes-Lamothe R,
    4. Sherratt D
    (2012) The Escherichia coli SMC complex, MukBEF, shapes nucleoid organization independently of DNA replication. J Bacteriol 194(17):4669–4676.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Danilova O,
    2. Reyes-Lamothe R,
    3. Pinskaya M,
    4. Sherratt D,
    5. Possoz C
    (2007) MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves. Mol Microbiol 65(6):1485–1492.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Gruber S,
    2. Errington J
    (2009) Recruitment of condensin to replication origin regions by ParB/SpoOJ promotes chromosome segregation in B. subtilis. Cell 137(4):685–696.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Gerdes K,
    2. Howard M,
    3. Szardenings F
    (2010) Pushing and pulling in prokaryotic DNA segregation. Cell 141(6):927–942.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Mohl DA,
    2. Gober JW
    (1997) Cell cycle-dependent polar localization of chromosome partitioning proteins in Caulobacter crescentus. Cell 88(5):675–684.
    OpenUrlPubMed
  41. ↵
    1. Toro E,
    2. Hong SH,
    3. McAdams HH,
    4. Shapiro L
    (2008) Caulobacter requires a dedicated mechanism to initiate chromosome segregation. Proc Natl Acad Sci USA 105(40):15435–15440.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Vallet-Gely I,
    2. Boccard F
    (2013) Chromosomal organization and segregation in Pseudomonas aeruginosa. PLoS Genet 9(5):e1003492.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Yamaichi Y,
    2. Fogel MA,
    3. Waldor MK
    (2007) par genes and the pathology of chromosome loss in Vibrio cholerae. Proc Natl Acad Sci USA 104(2):630–635.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. Wang X,
    2. Tang OW,
    3. Riley EP,
    4. Rudner DZ
    (2014) The SMC condensin complex is required for origin segregation in Bacillus subtilis. Curr Biol 24(3):287–292.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Gruber S,
    2. et al.
    (2014) Interlinked sister chromosomes arise in the absence of condensin during fast replication in B. subtilis. Curr Biol 24(3):293–298.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Ribeiro SA,
    2. et al.
    (2009) Condensin regulates the stiffness of vertebrate centromeres. Mol Biol Cell 20(9):2371–2380.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Marko JF
    (2009) Linking topology of tethered polymer rings with applications to chromosome segregation and estimation of the knotting length. Phys Rev E Stat Nonlin Soft Matter Phys 79(5 Pt 1):051905.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Vecchiarelli AG,
    2. Mizuuchi K,
    3. Funnell BE
    (2012) Surfing biological surfaces: Exploiting the nucleoid for partition and transport in bacteria. Mol Microbiol 86(3):513–523.
    OpenUrlCrossRefPubMed
  49. ↵
    1. Vecchiarelli AG,
    2. Hwang LC,
    3. Mizuuchi K
    (2013) Cell-free study of F plasmid partition provides evidence for cargo transport by a diffusion-ratchet mechanism. Proc Natl Acad Sci USA 110(15):E1390–E1397.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    1. Murray H,
    2. Errington J
    (2008) Dynamic control of the DNA replication initiation protein DnaA by Soj/ParA. Cell 135(1):74–84.
    OpenUrlCrossRefPubMed
  51. ↵
    1. Youngren B,
    2. Nielsen HJ,
    3. Jun S,
    4. Austin S
    (2014) The multifork Escherichia coli chromosome is a self-duplicating and self-segregating thermodynamic ring polymer. Genes Dev 28(1):71–84.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Veening JW,
    2. Murray H,
    3. Errington J
    (2009) A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilis. Genes Dev 23(16):1959–1970.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    1. Harwood CR,
    2. Cutting SM
    (1990) Molecular Biological Methods for Bacillus (Wiley, New York).
  54. ↵
    1. Grossman AD,
    2. Losick R
    (1988) Extracellular control of spore formation in Bacillus subtilis. Proc Natl Acad Sci USA 85(12):4369–4373.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    1. Wu LJ,
    2. Errington J
    (1994) Bacillus subtilis SpoIIIE protein required for DNA segregation during asymmetric cell division. Science 264(5158):572–575.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    1. Griffith KL,
    2. Grossman AD
    (2008) Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP. Mol Microbiol 70(4):1012–1025.
    OpenUrlPubMed
PreviousNext
Back to top
Article Alerts
Email Article

Thank you for your interest in spreading the word on PNAS.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Bacillus subtilis chromosome organization oscillates between two distinct patterns
(Your Name) has sent you a message from PNAS
(Your Name) thought you would like to see the PNAS web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Oscillating chromosome organization patterns
Xindan Wang, Paula Montero Llopis, David Z. Rudner
Proceedings of the National Academy of Sciences Sep 2014, 111 (35) 12877-12882; DOI: 10.1073/pnas.1407461111

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Request Permissions
Share
Oscillating chromosome organization patterns
Xindan Wang, Paula Montero Llopis, David Z. Rudner
Proceedings of the National Academy of Sciences Sep 2014, 111 (35) 12877-12882; DOI: 10.1073/pnas.1407461111
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Mendeley logo Mendeley

Article Classifications

  • Biological Sciences
  • Microbiology

See related content:

  • Bacterial chromosome dynamics
    - Aug 25, 2014
Proceedings of the National Academy of Sciences: 111 (35)
Table of Contents

Submit

Sign up for Article Alerts

Jump to section

  • Article
    • Abstract
    • Results
    • Discussion
    • Materials and Methods
    • Acknowledgments
    • Footnotes
    • References
  • Figures & SI
  • Info & Metrics
  • PDF

You May Also be Interested in

Smoke emanates from Japan’s Fukushima nuclear power plant a few days after tsunami damage
Core Concept: Muography offers a new way to see inside a multitude of objects
Muons penetrate much further than X-rays, they do essentially zero damage, and they are provided for free by the cosmos.
Image credit: Science Source/Digital Globe.
Water from a faucet fills a glass.
News Feature: How “forever chemicals” might impair the immune system
Researchers are exploring whether these ubiquitous fluorinated molecules might worsen infections or hamper vaccine effectiveness.
Image credit: Shutterstock/Dmitry Naumov.
Venus flytrap captures a fly.
Journal Club: Venus flytrap mechanism could shed light on how plants sense touch
One protein seems to play a key role in touch sensitivity for flytraps and other meat-eating plants.
Image credit: Shutterstock/Kuttelvaserova Stuchelova.
Illustration of groups of people chatting
Exploring the length of human conversations
Adam Mastroianni and Daniel Gilbert explore why conversations almost never end when people want them to.
Listen
Past PodcastsSubscribe
Horse fossil
Mounted horseback riding in ancient China
A study uncovers early evidence of equestrianism in ancient China.
Image credit: Jian Ma.

Similar Articles

Site Logo
Powered by HighWire
  • Submit Manuscript
  • Twitter
  • Facebook
  • RSS Feeds
  • Email Alerts

Articles

  • Current Issue
  • Special Feature Articles – Most Recent
  • List of Issues

PNAS Portals

  • Anthropology
  • Chemistry
  • Classics
  • Front Matter
  • Physics
  • Sustainability Science
  • Teaching Resources

Information

  • Authors
  • Editorial Board
  • Reviewers
  • Subscribers
  • Librarians
  • Press
  • Cozzarelli Prize
  • Site Map
  • PNAS Updates
  • FAQs
  • Accessibility Statement
  • Rights & Permissions
  • About
  • Contact

Feedback    Privacy/Legal

Copyright © 2021 National Academy of Sciences. Online ISSN 1091-6490