Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis - PubMed (original) (raw)

Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis

Jerod L Ptacin et al. Nat Struct Mol Biol. 2008 May.

Abstract

In prokaryotes, the transfer of DNA between cellular compartments is essential for the segregation and exchange of genetic material. SpoIIIE and FtsK are AAA+ ATPases responsible for intercompartmental chromosome translocation in bacteria. Despite functional and sequence similarities, these motors were proposed to use drastically different mechanisms: SpoIIIE was suggested to be a unidirectional DNA transporter that exports DNA from the compartment in which it assembles, whereas FtsK was shown to establish translocation directionality by interacting with highly skewed chromosomal sequences. Here we use a combination of single-molecule, bioinformatics and in vivo fluorescence methodologies to study the properties of DNA translocation by SpoIIIE in vitro and in vivo. These data allow us to propose a sequence-directed DNA exporter model that reconciles previously proposed models for SpoIIIE and FtsK, constituting a unified model for directional DNA transport by the SpoIIIE/FtsK family of AAA+ ring ATPases.

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Figures

Figure 1

Architecture of SpoIIIE and FtsK monomers and models for DNA translocation directionality. (a) Schematic depicting the architecture of the SpoIIIE and FtsK monomers (above and below, respectively), showing the N-terminal transmembrane domain (blue), the central linker domain (white) and the motor domain consisting of α, β and γ subdomains (red). (b) Sequence-directed model for DNA translocation directionality of FtsK. Above left, FtsK moves along DNA in the direction of the white arrow. The encounter of FtsK with KOPS in the permissive direction (rightward-pointing red arrowheads) does not affect the translocation direction (middle left). FtsK can reverse translocation direction (below left) when encountering KOPS in the nonpermissive orientation (leftward-pointing red arrowheads). KOPS are highly skewed in the chromosome of E. coli and switch strand and orientation at dif (right). (c) Simple exporter model for DNA translocation directionality by SpoIIIE. In this model, the relative abundance of SpoIIIE (red circle, arrows indicating its relative orientation) in the mother cell or other cell-specific factors favor the assembly of a unidirectional DNA exporter in the mother cell that determines the directionality of transfer (left). Expression of SpoIIIE in the forespore after septation leads to DNA transfer into the mother cell (right).

Figure 2

SpoIIIE-γ is necessary for efficient sporulation but does not affect motor function. (a) Log of viable spore titers for wild-type (red), SpoIIIE null (white), SpoIIIE★ (orange) and SpoIIIE★-Δγ (green) cells. Error bars indicate s.d. (b) Timelapse microscopic images of wild-type (above), SpoIIIE★ (middle) and SpoIIIE★-Δγ (below) cells. Membrane images (red) at the left are enhanced to indicate the location and orientation of the cell in each frame, and the forespore and mother cell are indicated (FS and MC, respectively). A time-lapse image series is shown, with arrows indicating the forespore DNA (green). The scale bar of 1 μm is shown. Plots to the right of each series show the normalized forespore intensity versus time. (c) Normalized DNA fluorescence intensity in the forespore versus that in the mother cell from time-lapse fluorescence images of SpoIIIE★ strains permits quantification of the percentage of DNA translocated into the forespore as a function of time. (d) Normalized DNA fluorescence intensity traces of individual cells (different colors shown) of the SpoIIIE★-Δγ strain. (e) In vivo velocities of DNA translocation for SpoIIIE (red), SpoIIIE★ (orange) and SpoIIIE★-Δγ (green) obtained from DNA fluorescence intensity traces. Error bars indicate s.d.

Figure 3

Identification and characterization of a SpoIIIE recognition sequence (SRS). (a) Schematic depicting the orientation of SRS6 sequences (blue arrowheads) along the B. subtilis chromosome (circle). The replication origin (oriC), the dif sequence and the polar localization region (PLR) are indicated. Gray area marks the region of the chromosome initially trapped by the septum before DNA translocation by SpoIIIE commences. (b) Schematic depicting the magnetic tweezers setup for DNA sequence recognition. A naked double-stranded DNA molecule (black helix) is tethered between a glass slide and magnetic particle. A repeat of three SRS candidates to be tested (blue arrowheads) is located near the top, 1.1 μm from the bead and 2.9 μm from the glass surface. Tension in the DNA is introduced using magnets (red triangles). SpoIIIEC-induced DNA looping shortens the DNA extension, monitored by the change in height of the magnetic particle using an inverted objective (white rhombus). Specific interactions between SpoIIIEC and SRS are observed as pauses and translocation reversals at extensions corresponding to the location of the tandem repeat of three SRS sequences (3 × SRS). (c) Representative trace of the change in DNA extension due to single-molecule SpoIIIEC activity on a DNA molecule containing a 3 × SRS6 at the test location (black line). (d) Schematic of DNA triplex displacement assay. SRS repeats (arrowheads) in the nonpermissive or the permissive orientations are located on a linear duplex DNA between a 21-bp triplex (jagged line) and a 3-kb ‘antenna’ region. SpoIIIE (blue sphere) often binds within the antenna region because of its relative length. Translocation of SpoIIIE into the nonpermissive SRS region frequently reverses SpoIIIE, protecting the triplex (above). SpoIIIE translocation into the permissive SRS region leads to displacement of the triplex (below). (e) SpoIIIEC triplex displacement reactions on DNA substrates with permissive (green circles) or nonpermissive (red squares) SRS6 orientations. The percentage of free triplex is plotted as a function of time, and error bars indicate s.d. Data were normalized for the initial percentage of free triplex and fit to an exponential function. The ratio of displacement on nonpermissive versus permissive substrates is 2.5 ± 0.5. (f) Step plot indicating the locations and orientations of SRS6 along the B. subtilis genome (see text). Cumulative skew is shown as a function of chromosomal location (Supplementary Methods). The replication origin (oriC), the dif site and the PLR are indicated. Gray area marks the region of the chromosome initially trapped by the septum before DNA segregation by SpoIIIE commences.

Figure 4

γ-domains are modular and can be switched between species to confer altered DNA sequence specificities. (a) Schematic depicting the architecture of the SpoIIIE-SK chimera containing the γ-domain of FtsK (dark red) fused to the motor domain of SpoIIIE (light red). (b) Representative trace of SpoIIIEC-SK chimera-induced changes in DNA extension versus time in the magnetic tweezers on a DNA substrate containing a triple repeat of KOPS (3 × SRS1) at the test location (black line). (c) SpoIIIEC-SK chimera DNA triplex displacement reactions on substrates containing either a triple KOPS sequence in permissive (green circles) or nonpermissive (red squares) orientations. Error bars indicate s.d. Data were normalized for the initial percentage of free triplex and fit to an exponential function. Ratio of displacement on permissive versus nonpermissive substrates is 1.8 ± 0.25. (d) Log of heat-resistant spore titers for wild-type SpoIIIE★ (orange), SpoIIIE★-Δγ (green) and SpoIIIE★-SK chimera (red) strains. Error bars indicate s.d. (e) Normalized DNA fluorescence intensity traces of individual SpoIIIE★-SK cells (different colors shown). Velocities of DNA translocation in vivo were 520 ± 100 bp s−1 (n = 16), similar to those of SpoIIIE★.

Figure 5

Cell-specific GFP tagging indicates that SpoIIIE-Δγ assembles on both sides of the sporulation septum with equal frequency. Cartoons show the experimental setup (right). FosLZ–GFP (green circles) was expressed after septation in the forespore or mother-cell compartments in strains expressing SpoIIIE–JunLZ or SpoIIIE–Δγ–JunLZ (white circles). The location of SpoIIIE complexes was observed by the appearance of a GFP focus (green cluster) at the septal midpoint. Fluorescence microscopic images show a membrane and GFP overlay (left), a DNA and GFP overlay (center) and GFP only (right). GFP foci are indicated by white arrowheads. (a) SpoIIIE assembles only on the mother-cell side of the septum during DNA translocation. SpoIIIE–JunLZ foci were observed on the mother-cell side of the septum in 48% of the cells (n = 191, above), whereas no SpoIIIE–JunLZ foci were observed in the forespore compartment (n = 182, below). (b) SpoIIIE–Δγ assembles on both sides of the septal membrane with equal frequency. Assembly of SpoIIIE–Δγ–JunLZ foci was observed in the mother cell (53%, n = 210, above) and forespore (47%, n = 232, below) sides of the septum.

Figure 6

Model for SpoIIIE sequence-directed DNA export during sporulation. (a) Schematic of a sporulating B. subtilis cell (gray) at the onset of DNA translocation, in which each chromosomal arm (black ribbon) is bound by two opposing active unidirectional complexes (green) that assemble on each side of the division septum (brown disc). Below, an enlarged view of a single chromosomal arm (helix), division septum (gray) and SRS sequences (blue triangles) are represented. Black arrows depict the overall direction of DNA transport. We assume that during translocation DNA is transported in the direction from SpoIIIE-β to SpoIIIE-α; (b) SpoIIIE-γ–mediated interactions with nonpermissive SRS in the forespore leads to the inactivation of the forespore complex (red). (c) The inactivation of the forespore SpoIIIE complex converts the bidirectional channel into a DNA exporter, leading to unidirectional DNA transport into the forespore.

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Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis - PubMed (original) (raw)