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, 9 (1), 8315

Replication Termination Without a Replication Fork Trap

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Replication Termination Without a Replication Fork Trap

Elisa Galli et al. Sci Rep.

Abstract

Bacterial chromosomes harbour a unique origin of bidirectional replication, oriC. They are almost always circular, with replication terminating in a region diametrically opposite to oriC, the terminus. The oriC-terminus organisation is reflected by the orientation of the genes and by the disposition of DNA-binding protein motifs implicated in the coordination of chromosome replication and segregation with cell division. Correspondingly, the E. coli and B. subtilis model bacteria possess a replication fork trap system, Tus/ter and RTP/ter, respectively, which enforces replication termination in the terminus region. Here, we show that tus and rtp are restricted to four clades of bacteria, suggesting that tus was recently domesticated from a plasmid gene. We further demonstrate that there is no replication fork system in Vibrio cholerae, a bacterium closely related to E. coli. Marker frequency analysis showed that replication forks originating from ectopic origins were not blocked in the terminus region of either of the two V. cholerae chromosomes, but progressed normally until they encountered an opposite fork. As expected, termination synchrony of the two chromosomes is disrupted by these ectopic origins. Finally, we show that premature completion of the primary chromosome replication did not modify the choreography of segregation of its terminus region.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Tus-related homologs in bacteria. Phylogenetic tree of the proteins containing the Tus HHM profile (PF05472) found in complete genome of UniProt database and named as the sequenced species. Only three monophyletic groups of proteins were considered as “resident” as they present a distribution congruent with that of the species containing these Tus proteins established with a DnaABEX-based phylogenetic tree. Most Enterobacteriales (only a subset is indicated) highlighted in purple ①, the Pseudoalteromonas in which tus is present on the second chromosome highlighted in blue ②, and all the Aeromonadales, except Tolumonas aurensis TA4, highlighted in green ③. By opposition, the other tus-related genes, which do not meet the residency criteria explained in the main text, are considered as “mobile” genes. Some are plasmid-borne (highlighted in yellow) and other are located on chromosomes (not highlighted). Scale bar represents 0.1 substitutions per site; Bootstrap scores of domestication branches are indicated on the figure.
Figure 2
Figure 2
MFA of the two-chromosome strains: V. cholerae N16961 (EPV50; A) and derivatives containing ectopic oriC1 ((EGV140; (B) and EGV111; C)). Marker frequencies (grey dots after trimming (see Supp. Methods) and normalisation on the total number of reads) are represented in Log2 as a function of the genome position. The oriC1 or oriC2 of chr1 or chr2, respectively, are indicated at each extremity. Position of dif1, dif2, ectopic origin if applicable (oriL3 or oriR4), crtS, the different mp (origins mid-point) are indicated. The lowest point on chr1 was set to “1” in such a way that log2(1) = “0” and all data were normalized to this point. The curve fitting the marker frequency data (see Supp. Methods for MFA method) are indicated by either a blue or a red line for chr1 and chr2, respectively. They define the forks convergence points (fcp), indicated under the data. On the left side of the marker frequency data, a scheme representing the program of replication of chr1 is indicated on the circular map of the different strains. The program of replication of chr2 is represented only for EPV50 as it is not modified in EGV140 and EGV111. The program of replication deduced from the MFA is as follow: plain grey line corresponds to the wild-type direction of fork progression and the dashed grey line to the reverse direction of fork progression. The distance between fcp and its mp (noted fcp-mp) is indicated in % of the replicon fraction, oriented from the first origin encounters in the clock-wise direction.
Figure 3
Figure 3
MFA of the mono-chromosome strains: V. cholerae MCH1 (A) and derivatives containing ectopic oriC1 (EGV369; (B) and EGV366; (C)). See Fig. 2 legend, adapted for the fusion between chr1 and chr2 (deleting dif1 region and oriC2 region).
Figure 4
Figure 4
Duplication and choreography of dif1 locus in WT and ectopic oriC1-containing derivatives. (A) Proportion of cells with 2 dif1-foci according to the cell length (cell size intervals of 0.1 µm). Population cell size is represented in the inset. (B) Choreography of dif1 in EGV360 (top panel), in EGV362 (middle panel) and in EGV361 (bottom panel). The relative positioning of dif1 along the cell length axis is in function of the cell length (cell size intervals of 0.1 µm); For each size interval, the median positioning of dif1 among the cells with 1 dif1- focus (plain dot) and the pair of median dif1 positioning among the cells with 2 dif1-foci (bi-coloured dot) are indicated (when proportion exceed 5% as shown by the dashed line in (A). The size of the dots reflects qualitatively the proportion between the 1- and the 2-dif1 cells among each size interval. (0: new pole; 1: old pole). For each strain, 2500 cells minimum were analysed.

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