Simultaneous imaging of SeqA and the Ter region.

<p>(<b>A</b>) Cell cycle diagram of cells (SF163), with tagged SeqA protein (SeqA-YFP) and Ter region (FROS), obtained by flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone.0110575.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone.0110575.s006" target="_blank">Table S1</a>). Cells were grown in glucose-CAA medium with a doubling time (τ) of 65 minutes. Initiation of replication at four origins occurred at age a_i = 39 minutes (average for the population), and the replication period (C-period) lasted for about 83 minutes. The C-period is indicated as a black line, and the D-period is shown as a stippled line (<b>B</b>) Representative images from snapshot fluorescence imaging shows formation of SeqA(pseudo-colored red) and Ter (pseudo-colored green) foci. N = 232 cells. (<b>C</b>) Schematic drawing of DNA and foci below the images, SeqA as red dot, Ter region as green dot, origin as black dot, DNA replicated by old forks in grey, DNA to be replicated by old forks in black and DNA replicated by new forks in blue. The placement of origins and DNA is in this illustration hypothetical and simplified, based on observed positioning of SeqA and Ter region in fluorescence images in addition to cell cycle parameters from 3A.</p

<i>Escherichia coli</i> SeqA Structures Relocalize Abruptly upon Termination of Origin Sequestration during Multifork DNA Replication

<div><p>The <i>Escherichia coli</i> SeqA protein forms complexes with new, hemimethylated DNA behind replication forks and is important for successful replication during rapid growth. Here, <i>E. coli</i> cells with two simultaneously replicating chromosomes (multifork DNA replication) and YFP tagged SeqA protein was studied. Fluorescence microscopy showed that in the beginning of the cell cycle cells contained a single focus at midcell. The focus was found to remain relatively immobile at midcell for a period of time equivalent to the duration of origin sequestration. Then, two abrupt relocalization events occurred within 2–6 minutes and resulted in SeqA foci localized at each of the cell’s quarter positions. Imaging of cells containing an additional fluorescent tag in the origin region showed that SeqA colocalizes with the origin region during sequestration. This indicates that the newly replicated DNA of first one chromosome, and then the other, is moved from midcell to the quarter positions. At the same time, origins are released from sequestration. Our results illustrate that newly replicated sister DNA is segregated pairwise to the new locations. This mode of segregation is in principle different from that of slowly growing bacteria where the newly replicated sister DNA is partitioned to separate cell halves and the decatenation of sisters a prerequisite for, and possibly a mechanistic part of, segregation.</p></div

Model of SeqA relocalization events.

<p>A schematic drawing to illustrate how new and unreplicated DNA may change places during the SeqA relocalization events reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone-0110575-g001" target="_blank">Figure 1</a>. (<b>A</b>) Images in red square (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone-0110575-g001" target="_blank">Figure 1C</a>) showing SeqA at the end of the period at midcell (t min), the first relocalization to one cell quarter (at t+1 min), and the second relocalization to the other cell quarter (at t+2 min). (<b>B</b>) Illustration of the position of SeqA and newly replicated DNA of two chromosomes during the period at midcell (cell number one), after the movement of first new and unreplicated DNA of the lower chromosome (cell number two), and then the upper chromosome (cell number three). It is here suggested that origins in sequestration are part of the midcell SeqA structures. The SeqA foci are illustrated as green dots, the origin foci as red squares, unreplicated DNA as a black line and newly replicated DNA as a grey line. The position of the unreplicated DNA is simplified and the Ter region localization at midcell is not included.</p

Live-cell fluorescence imaging of SeqA-YFP during rapid growth.

<p>Cells with the <i>YFP</i> gene inserted at the C-terminal end of the chromosomal <i>seqA</i> gene (SF128) were grown at 28°C on an agarose pad containing 1% glucose-CAA. The agarose pad was attached to a microscopy slide, and images were recorded every 1 minute over a 40 minutes period. (<b>A</b>) Cell cycle diagram with parameters obtained by flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone.0110575.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone.0110575.s006" target="_blank">Table S1</a>). The replication period (C-period) spanned about one doubling time (τ = 66 min). The origin sequestration period is shown in purple as part of the black line that indicates the C-period. Initiation of replication occurred at age a_i = 2 minutes (average for the population). The D-period is shown as a stippled line. Numbers presented in the diagram are average of four independent experiments. Schematic drawings of cells are shown above the diagram to illustrate DNA content, numbers of origins, numbers of replication forks and replication fork progression at different stages of the cell cycle (the drawings are not indicative of chromosome positioning or organization patterns). Chromosomes are shown as black lines and origins as black dots. (<b>B</b>) Histogram showing numbers of SeqA foci per cell in categories I–IV representing the progression along the cell cycle (see text for description of categories). Representative illustrations of the cells with SeqA foci (as green dots) within each category are shown above the histogram. (<b>C</b>) Time-lapse series of a representative cell from category I with one SeqA focus at midcell. Scale bar is 1 µm. Numbers on pictures indicate time after start of imaging. (<b>D</b>) Analysis of SeqA dynamics during live-cell imaging. The positions of the SeqA foci along the cell length were plotted as a function of time for the cell from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone-0110575-g001" target="_blank">Figure 1C</a>. The average positions of SeqA foci relative to the cell pole (obtained from six cells from category I) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone.0110575.s003" target="_blank">Figure S3</a>. The red box in (<b>C</b>) and (<b>D</b>) indicates relocalization of SeqA from midcell towards the quarter positions at 14–16 minutes after start of imaging.</p

Period of hemimethylation at <i>oriC</i>.

<p>Period of hemimethylation at <i>oriC</i>.</p

Numbers of cells in which SeqA and origin region are colocalized (yellow foci) or not (red/green foci).

<p>Numbers of cells in which SeqA and origin region are colocalized (yellow foci) or not (red/green foci).</p

Categories of cells (SF128) with characteristic features from snapshot imaging (Figure 1B).

<p>Categories of cells (SF128) with characteristic features from snapshot imaging (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone-0110575-g001" target="_blank">Figure 1B</a>).</p

Simultaneous imaging of SeqA and the origin region.

<p>(<b>A</b>) Cell cycle diagram of cells (SF131), with fluorescently tagged SeqA protein (SeqA-YFP) and origin region (FROS), obtained by flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone.0110575.s001" target="_blank">Figure S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110575#pone.0110575.s006" target="_blank">Table S1</a>). Cells were grown in glucose-CAA medium with a doubling time (τ) of 66 minutes. Initiation of replication occurred at age a_i = 0 minutes (average for the population), and the replication period (C-period) lasted for about 72 minutes. The origin sequestration period is shown in purple as part of the black line that indicates the C-period. The D-period is shown as a stippled line. (<b>B</b>) Snapshot fluorescence imaging shows formation of discrete SeqA (pseudo-colored red) and origin (pseudo-colored green) foci. Occurrence of yellow foci indicates colocalization of the SeqA protein and origin region within the resolution of the microscope. The percentage of cells with yellow foci is indicated. N = 476 cells.</p

The DnaA Protein Is Not the Limiting Factor for Initiation of Replication in <i>Escherichia coli</i>

<div><p>The bacterial replication cycle is driven by the DnaA protein which cycles between the active ATP-bound form and the inactive ADP-bound form. It has been suggested that DnaA also is the main controller of initiation frequency. Initiation is thought to occur when enough ATP-DnaA has accumulated. In this work we have performed cell cycle analysis of cells that contain a surplus of ATP-DnaA and asked whether initiation then occurs earlier. It does not. Cells with more than a 50% increase in the concentration of ATP-DnaA showed no changes in timing of replication. We suggest that although ATP-DnaA is the main actor in initiation of replication, its accumulation does not control the time of initiation. ATP-DnaA is the motor that drives the initiation process, but other factors will be required for the exact timing of initiation in response to the cell’s environment. We also investigated the <i>in vivo</i> roles of <i>datA</i> dependent DnaA inactivation (DDAH) and the DnaA-binding protein DiaA. Loss of DDAH affected the cell cycle machinery only during slow growth and made it sensitive to the concentration of DiaA protein. The result indicates that compromised cell cycle machines perform in a less robust manner.</p></div

Strains and plasmids.

<p>Strains and plasmids.</p