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

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

Matthias Bruhn et al. Front Microbiol.

Abstract

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.

Keywords: DNA replication; cholera; multipartite genome; pathogens; plasmid; replication initiation; secondary chromosome.

Figures

FIGURE 1
FIGURE 1
Genetic maps of V. cholerae NSCV1 and NSCV2 chromosomes depicting fusion junctions and other genomic features. (A) The fused chromosomes of strain NSCV1 (left) and NSCV2 (right) are shown with the original Chr1 and Chr2 in black and blue parts of the ring, respectively. The origins are indicated by red dots and the crtS sites by green stars. (B,C) Inversion analysis of V. cholerae NSCV1 and NSCV2 chromosomes using Smash tool (Pratas et al., 2015). The genome sequences were analyzed for sequence homology with the two-chromosome genome of strain N16961 as reference (accession numbers NC002505 and NC002506). Regions with identical information content are marked by the same color. Inverted regions are indicated by waved lines. White areas represent unmatched regions or Chr2.
FIGURE 2
FIGURE 2
Single nucleotide polymorphism (SNP) analysis of replication origins of V. cholerae strains NSCV1 and NSCV2 in comparison to N16961. Genes are indicated as black arrows, RctB binding iterons in dark gray (11 mer iteron) and light gray (12 mer iteron), DnaA boxes in blue and SNPs as stars. The figure is not drawn to scale. Reference sequence: N16961 (Accession # NC002505 and NC002506). Apart from the highlighted SNPs, the sequences are identical. (A) Alignment of ori1. (B) Alignment of ori2.
FIGURE 3
FIGURE 3
Marker frequency analysis (MFA) of V. cholerae NSCV1 (A) and NSCV2 (B) to assess origin activities. Profiles of genome-wide copy numbers based on Illumina sequencing and read mapping. Gray dots represent log numbers of normalized reads as mean values for 1 kbp windows relative to the stationary phase sample. The genome position is shown as the distance from ori1. Vertical dotted black lines mark the locations of replication origins and the crtS site. The solid black lines represent the fitting of regression lines and the green line corresponds to the Loess regression (F = 0.05). Maxima are highlighted by red and minima as blue dots. Plots of biological replicates are shown in Supplementary Figure S1.
FIGURE 4
FIGURE 4
Differential interference contrast (DIC) microscopic images of cells of various V. cholerae strains growing in exponential phase. (A–C) N16961 represents the typical 2 chromosome V. cholerae and NSCV1 and NSCV2 are Chr1 and Chr2 fusion strains.
FIGURE 5
FIGURE 5
Marker frequency analysis of engineered single chromosome V. cholerae strains VC61 (A) and VC62 (B). Left panel: Genomic maps of VC61 (analogous to NSCV1) and VC62 (analogous to NSCV2) showing the respective locations of ori1, ori2, and crtS. Right panel: Profiles of genome-wide copy numbers based on Illumina sequencing. Gray dots represent log numbers of normalized reads as mean values for 1 kbp windows relative to the stationary phase sample. Vertical dotted black lines mark the locations of replication origins of replication and the crtS sites. The solid black lines represent the fitting of regression lines and the green line corresponds to the Loess regression (F = 0.05). Maxima are highlighted by red and minima as blue dots. Plots of biological replicates are shown in Supplementary Figure S1.
FIGURE 6
FIGURE 6
Testing Dam methylation sensitivities of genomic DNAs of strains V. cholerae NSCV1 and NSCV2. Restriction digestion of 1 μg of genomic DNA using the indicated enzymes was carried out and the various strains are as follows: 1. E. coli MG1655; 2. E. coli MG1655Δdam; 3 and 4. V. cholerae NSCV1; 5 and 6. V. cholerae NSCV2. DpnI cleaves methylated GATC sequences. DpnII cleaves only unmethylated GATC sequences. Sau3AI cleaves GATCs independent of the methylation state. DNA cleavage is evident from the disappearance of high molecular weight band and appearance of low molecular weight streak of DNA.
FIGURE 7
FIGURE 7
crtS sites of NSCV strains are functional. (A) An alignment of crtS sites from 13 sequenced Vibrio genomes was used to calculate a WebLogo (top panel). The height of the nucleotides in each position represents the measure of conservation. All positions with 100% sequence conservation are conserved in the crtS sequences of NSCV1 and NSCV2 as well (indicated in bold, lower panel) (Kemter et al., 2018). (B) Marker frequency analysis of V. cholerae strain VC71 lacking a functional crtS site. Gray dots represent log numbers of normalized reads as mean values for 1 kbp windows relative to the stationary phase sample. The genome position is shown as the distance from ori1. Vertical dotted black lines mark the locations of replication origins of replication and the crtS sites. The solid black lines represent the fitting of regression lines and the green line corresponds to the Loess regression (F = 0.05). Maxima are highlighted by red and minima as blue dots. Plots of biological replicates are shown in Supplementary Figure S1. (C) crtS sites of NSCV1 and NSCV2 increase the copy number of ori2-based mini-chromosomes in E. coli. E. coli strains harboring the ori2-based mini-chromosome synVicII and chromosomal insertions of crtS from different V. cholerae strains as indicated were grown in LB medium with 500 μg/ml ampicillin in a 96-well plate at 37°C. As strains with a lower replicon copy number have a longer lag period to initiate growth, the value 1 divided by the time to reach an OD600 ≥ 0.1 was used as measure for the replicon copy number. Values are the mean of three biological replicates with indicated standard deviation. Growing the strains in standard concentrations of ampicillin did not show any difference between wild type and crtS carrying strains as expected (data not shown).
FIGURE 8
FIGURE 8
Marker frequency analysis of V. cholerae strain VC73. Left panel: Genomic map of VC73 showing the locations of ori1, ori2, and crtS. Right panel: Profile of genome-wide copy numbers. Gray dots represent log numbers of normalized reads as mean values for 1 kbp windows relative to the stationary phase sample. The genome position is shown as the distance from ori1. Vertical dotted black lines mark the locations of replication origins of replication and the crtS sites. The solid black lines represent the fitting of regression lines and the green line corresponds to the Loess regression (F = 0.05). Maxima are highlighted by red and minima as blue dots. Plots of biological replicates are shown in Supplementary Figure S1.

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