Figure 1.
Map of the wild type and XL151 chromosomes.
The positions of Inv(dif-sp5) inversion endpoints (the sp5 and the zdd347 loci) and the loci used for parS insertions (aidB, crl, ydeQ and ydeV) are indicated. Coordinates are in kb. The black flags are the replication terminators (Ter sites with corresponding letter from A to J). The grey arrowheads and numbers represent the polarity (orientation of KOPS motifs) of replichore 1 and 2, respectively, and the grey heavy line the Ter macrodomain. PJ: polarity junction that contains the dif site (the black and white square); oriC: origin of replication.
Figure 2.
Phenotype of the XL151 strain.
A) XL151 was grown in M9 broth at 30°C (permissive conditions; closed circles) and growth was followed by plating in permissive conditions (cfu, left scale). At the indicated time, one half of the culture was shifted to 42°C (non-permissive conditions; red triangles). The fraction of dead cells was followed in both culture using live and dead staining (300 cells per count; right scale; black bars: permissive conditions; red bars: non-permissive conditions). A micrograph of XL151 stained with Live/dead staining (Materials and Methods) after incubation 5 hours in non-permissive conditions is shown (dead cells: red; Live cells: green; examples are indicated with the red and green dots, respectively). B) Top: micrographs of wt (left) and XL151 (right) after 1 hour at 42°C (non-permissive conditions). Example of cells counted as single (yellow dots) and double cells (red dots) are shown. Bottom: cells with one nucleoid (single cells), cells with two apparently segregated nucleoids (harbouring or not harbouring a division septum; double cells) and abnormal cells (filaments and chains) were counted in permissive conditions (30°C) and after 1 hour in non permissive conditions (42°C) (300 cells per count). C) The cell size distribution of wt (left) and XL151 (right) was determined by flow cytometry in different conditions: after overnight growth in permissive conditions (stationary phase, white); in exponential growth in permissive conditions (growth 30°C, pale grey); after one hour incubation in non-permissive conditions (42°C-1H, dark grey) and 1 hour after addition of cephalexin in permissive conditions (dash line). Size is in mm (x-axis). Quantifications of the fractions of cell under 4 µm, from 4 µm to 7 µm and over 7 µm are presented (see also Table S1).
Table 1.
Cell cycle parameters of wt and XL151 strains.
Figure 3.
Replication in wild type and XL151.
A) DNA content distribution of the wt (left) and XL151 (right) strains determined by flow cytometry after rifampicin/cephalexin run out (Material and methods) in stationary phase, exponential growth in permissive conditions (growth 30°C) and 1 hour in non-permissive conditions (42°C 1h). Chromosome equivalent are given for the different peaks. The average numbers of replication origins per cell in the three growth conditions is given below the graphs (see also Table S2). B) The C period of wt and XL151 cells growing under permissive conditions was measured using two techniques. 1the ori/ter ratio was determined by quantitative Southern blot hybridisation and the corresponding C period duration calculated. Data are the mean of three independent experiments with standard deviations (see Figure S1 for details). 2Data from flow cytometry analysis of the DNA content per cell after rifampicin treatment (Material and methods and Figure S1).
Figure 4.
Chromosome segregation patterns in wild type and XL151.
Top: representation of the wt and XL151 chromosomes with the replichore polarity junction (PJ), the termination zone (red zone) and the positions of oriC, dif and the parS insertions indicated. A to H: Intracellular localisation of the different parS insertions (indicated on the left) in wt (A to D) and XL151 (E to F). The dot plots represent the intracellular positioning of parS foci along the long axis of the cell (Y-axis with the cell poles, mid-cell and quarter positions indicated) as a function of cell length (X-axis) . Each plot represents data from 300 cells. Cell with one, two, three and four foci are respectively indicated in black, red, yellow and green. The histograms represent the proportion of cells with 1 focus (black), 2 (red), 3 (yellow) or 4 (green) foci. The Co-localisation Index (Ci) is obtained by dividing the mean locus copy number by the mean number of distinguishable fluorescent foci.
Figure 5.
Effect of the restoration of termination opposite oriC.
A) representation of the XL151 and the XL151 tus- chromosome with the replichore polarity junction (PJ), the termination zone (red zone) and the positions of oriC, dif and the parS insertion used indicated. B) intracellular positioning of the crl (left) and ydeQ (right) loci in XL151 tus-. same legend as Figure 4. C) Intracellular positioning of the different loci as a function of their position with respect to the replication termination zone. Cells were divided in 5 successive intervals from mid-cell to the pole and the fraction of foci in each interval is reported. Left: loci close to the termination zone. Cells with 2 foci are reported except for the ydeQ and ydeV loci for which cells with 1 focus are plotted. Right: loci far from the termination zone. Only cells with two foci are ploted. Data are from Figure 4 and 5B. The dashed grey line indicates the quarter position.
Table 2.
Doubling time of XL151 and derivatives.
Figure 6.
Depletion of FtsK was done in wt ftsKATP- and XL151 ftsKATP- strains containing a PBAD-ftsKwt allele on a plasmid. (A and B): Growth in presence of arabinose (0,2%) allows complementation of the ftsKATP- phenotype in both the wt ftsKATP- (A) and XL151 ftsKATP- (B) strains. (C and D): Depletion of FtsKwt was achieved by growing the cells 3 hours in liquid minimal medium containing 0.2% glucose at 30°C before microscopic observation. (C): wt ftsKATP-; (D) XL151 ftsKATP-. (E) Compilation of aberrantly shaped cells observed in XL151 ftsKATP- depleted for FtsKwt.