Functionality of Two Origins of Replication in Vibrio cholerae Strains With a Single Chromosome

Chromosomal inheritance in bacteria usually entails bidirectional replication of a single chromosome from a single origin into two copies and subsequent partitioning of one copy each into daughter cells upon cell division. However, the human pathogen Vibrio cholerae and other Vibrionaceae harbor two chromosomes, a large Chr1 and a small Chr2. Chr1 and Chr2 have different origins, an oriC-type origin and a P1 plasmid-type origin, respectively, driving the replication of respective chromosomes. Recently, we described naturally occurring exceptions to the two-chromosome rule of Vibrionaceae: i.e., Chr1 and Chr2 fused single chromosome V. cholerae strains, NSCV1 and NSCV2, in which both origins of replication are present. Using NSCV1 and NSCV2, here we tested whether two types of origins of replication can function simultaneously on the same chromosome or one or the other origin is silenced. We found that in NSCV1, both origins are active whereas in NSCV2 ori2 is silenced despite the fact that it is functional in an isolated context. The ori2 activity appears to be primarily determined by the copy number of the triggering site, crtS, which in turn is determined by its location with respect to ori1 and ori2 on the fused chromosome

Establishing a System for Testing Replication Inhibition of the Vibrio cholerae Secondary Chromosome in Escherichia coli

Regulators of DNA replication in bacteria are an attractive target for new antibiotics, as not only is replication essential for cell viability, but its underlying mechanisms also differ from those operating in eukaryotes. The genetic information of most bacteria is encoded on a single chromosome, but about 10% of species carry a split genome spanning multiple chromosomes. The best studied bacterium in this context is the human pathogen Vibrio cholerae, with a primary chromosome (Chr1) of 3 M bps, and a secondary one (Chr2) of about 1 M bps. Replication of Chr2 is under control of a unique mechanism, presenting a potential target in the development of V. cholerae-specific antibiotics. A common challenge in such endeavors is whether the effects of candidate chemicals can be focused on specific mechanisms, such as DNA replication. To test the specificity of antimicrobial substances independent of other features of the V. cholerae cell for the replication mechanism of the V. cholerae secondary chromosome, we establish the replication machinery in the heterologous E. coli system. We characterize an E. coli strain in which chromosomal replication is driven by the replication origin of V. cholerae Chr2. Surprisingly, the E. coli ori2 strain was not inhibited by vibrepin, previously found to inhibit ori2-based replication

Synchronous termination of replication of the two chromosomes is an evolutionary selected feature in Vibrionaceae

<div><p><i>Vibrio cholerae</i>, the causative agent of the cholera disease, is commonly used as a model organism for the study of bacteria with multipartite genomes. Its two chromosomes of different sizes initiate their DNA replication at distinct time points in the cell cycle and terminate in synchrony. In this study, the time-delayed start of Chr2 was verified in a synchronized cell population. This replication pattern suggests two possible regulation mechanisms for other <i>Vibrio</i> species with different sized secondary chromosomes: Either all Chr2 start DNA replication with a fixed delay after Chr1 initiation, or the timepoint at which Chr2 initiates varies such that termination of chromosomal replication occurs in synchrony. We investigated these two models and revealed that the two chromosomes of various Vibrionaceae species terminate in synchrony while Chr2-initiation timing relative to Chr1 is variable. Moreover, the sequence and function of the Chr2-triggering <i>crtS</i> site recently discovered in <i>V</i>. <i>cholerae</i> were found to be conserved, explaining the observed timing mechanism. Our results suggest that it is beneficial for bacterial cells with multiple chromosomes to synchronize their replication termination, potentially to optimize chromosome related processes as dimer resolution or segregation.</p></div

Synchronization of <i>Vibrio cholerae</i> by serine hydroxamate treatment.

<p><b>(A)</b> Growth of <i>V</i>. <i>cholerae</i> A1552 without (green) and with (red) addition of SHX. Cells were grown in AB Glu CAA medium in a 96-well plate at 37°C. Addition of 0.9 mg/ml SHX is indicated by the black arrow. <b>(B)</b> Density maps of flow cytometry data of DNA-stained <i>V</i>. <i>cholerae</i> A1552. Exponentially grown cells in AB Glu CAA were treated with SHX for 150 min. After washing SHX off, samples were taken every five minutes as indicated. Cells were fixed with ethanol and stained with SYTOX Green. The flow cytometry data of the samples was aligned to the corresponding standard and converted into density maps. Red indicates a high density of cells, while blue—no cells. <b>(C)</b> Quantification of DNA content after release from SHX. Given numbers are mean values of three biological replicates as described in <b>(B)</b>. Error bars show standard deviations.</p

Replication start and end control models.

<p>(<b>A</b>) Scheme of replication patterns of <i>Vibrio cholerae</i>, replication start and end control model. Circles represent chromosomes, arrows the length and timing of DNA replication. Black stands for Chr1, red for Chr2. Grey dashed lines show start and end of DNA replication of <i>V</i>. <i>cholerae</i> Chr2. Green dashed lines indicate the corresponding expected start and end of DNA replication of Chr2. (<b>B</b>) Scheme of expected MFA plots of exponentially growing cells. Black lines represent regression lines for values of Chr1, red lines of Chr2. Dashed lines as in (<b>A</b>).</p

The <i>crtS</i> is conserved among Vibrionaceae.

<p>(<b>A</b>) Sequence alignment of <i>crtS</i> sites of 13 species within the Vibrionaceae, including <i>Vibrio</i>, <i>Aliivibrio</i> and <i>Photobacterium</i>. The alignment was done with Clustal Omega [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007251#pgen.1007251.ref067" target="_blank">67</a>] and MView [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007251#pgen.1007251.ref068" target="_blank">68</a>]. Blue highlights indicate sequence similarities, while orange rectangles the parts belonging to coding sequences of genes, black arrows start and end of the <i>crtS</i> as described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007251#pgen.1007251.ref025" target="_blank">25</a>]. Green and purple arrows are for orientation in comparing subfigure A and B. (<b>B</b>) WebLogo of <i>crtS</i> from 114 species of Vibrionaceae published on NCBI. The heights of letters corresponds to their conservation within the group of sequences [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007251#pgen.1007251.ref069" target="_blank">69</a>].</p

Conservation of termination synchrony in Vibrionaceae.

<p>(<b>A-K</b>) Profile of genome-wide copy numbers based on Illumina sequencing. Grey dots represent log numbers of reads as mean values for 5 kbp windows. The genome position is shown, indicated by vertical dotted black lines, as the distance from <i>ori1</i> and <i>ori2</i>, respectively. The <i>crtS</i> is also marked by a vertical dotted black line. The solid black lines represent the fitting of two regression lines. Maxima are highlighted in yellow, minima in red and the <i>crtS</i> in green. Horizontal lines show log copy numbers of <i>ter1</i>, <i>ter2</i>, <i>ori2</i> (all black) and <i>crtS</i> (green). Plots of biological replicates are shown in supporting <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007251#pgen.1007251.s006" target="_blank">S6 Fig</a>. (<b>L</b>) Comparison of <i>ter1/2</i> copy number ratio (red dots) and the ratio of two-thirds of <i>ori1</i> copy number divided by <i>ori2</i> copy number (yellow dots). Mean values for both ratios are indicated.</p

Comparative genomics support the replication end control model.

<p>Grey dots represent the expected values (full correlation of both parameters) of 29 fully sequenced Vibrionaceae (Supporting <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007251#pgen.1007251.s008" target="_blank">S1 Table</a>), while black dots, the observed values. Red dots are the values of the strains further analyzed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007251#pgen.1007251.g007" target="_blank">Fig 7</a>. Linear equations and R² values are for regression of the observed values. A replichore is calculated as half of the corresponding chromosome, <i>ter1</i> is calculated as <i>ori1</i> + half of Chr1 (<b>A</b>) Examination of start control model. (<b>B</b>) Examination of end control model.</p