Cycle-specific replication of chromosomal and F-plasmid origins

Cooper, Stephen

doi:10.1016/s0378-1097(98)00177-3

Cited by 3 publications

(3 citation statements)

References 10 publications

(25 reference statements)

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“…Substantial effort has been applied toward elucidating when during the bacterial cell cycle individual plasmids are replicated (Cooper and Keasling, 1998; Bogan et al ., 2001), but less is known about the timing of plasmid segregation in relation to each other or other host cell cycle events. The results presented here, taken together with those of previous studies (Niki and Hiraga, 1997; Jensen and Shapiro, 1999b; Gordon and Wright, 2000; Hiraga, 2000; Pogliano et al ., 2001), indicate that different plasmids are not only spatially separated in the cell, but they segregate at different times relative to one another.…”

Section: Discussionsupporting

confidence: 78%

Compatible bacterial plasmids are targeted to independent cellular locations in Escherichia coli

Ho¹

2002

The EMBO Journal

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Targeting of DNA molecules to specific subcellular positions is essential for efficient segregation, but the mechanisms underlying these processes are poorly understood. In Escherichia coli, several plasmids belonging to different incompatibility groups (F, P1 and RK2) localize preferentially near the midcell and quartercell positions. Here we compare the relative positions of these three plasmids using fluorescence in situ hybridization. When plasmids F and P1 were localized simultaneously using differentially labeled probes, the majority of foci (approximately 75%) were well separated from each other. Similar results were found when we compared the subcellular localization of F with RK2, and RK2 with P1: regardless of the number of foci per cell or growth conditions, most of the foci (70-80%) were not in close proximity to one another. We also localized RK2 in Pseudomonas aeruginosa and Vibrio cholerae, and found that plasmid RK2 localization is conserved across bacterial species. Our results suggest that each plasmid has its own unique subcellular address, implying a mechanism for the stable co-existence of plasmids in which subcellular targeting plays a major role.

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Section: Discussionsupporting

confidence: 78%

Compatible bacterial plasmids are targeted to independent cellular locations in Escherichia coli

Ho¹

2002

The EMBO Journal

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“…In this respect the secondary chromosome of V. cholerae can be classified as a real chromosome because its replication is coordinated with the cell cycle [14]. However, it has to be mentioned that cell‐cycle specific replication might also apply to plasmids, although contradicting results have been found for example for the F plasmid [58–60]. It is important to note that although we assume cell cycle dependent replication of synVicII for the interpretation of copy numbers (Figs.…”

Section: Discussionmentioning

confidence: 99%

Synthetic secondary chromosomes in Escherichia coli based on the replication origin of chromosome II in Vibrio cholerae

Messerschmidt

Kemter

Schindler

et al. 2014

Biotechnology Journal

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Recent developments in DNA-assembly methods make the synthesis of synthetic chromosomes a reachable goal. However, the redesign of primary chromosomes bears high risks and still requires enormous resources. An alternative approach is the addition of synthetic chromosomes to the cell. The natural secondary chromosome of Vibrio cholerae could potentially serve as template for a synthetic secondary chromosome in Escherichia coli. To test this assumption we constructed a replicon named synVicII based on the replication module of V. cholerae chromosome II (oriII). A new assay for the assessment of replicon stability was developed based on flow-cytometric analysis of unstable GFP variants. Application of this assay to cells carrying synVicII revealed an improved stability compared to a secondary replicon based on E. coli oriC. Cell cycle analysis and determination of cellular copy numbers of synVicII indicate that replication timing of the synthetic replicon in E. coli is comparable to the natural chromosome II (ChrII) in V. cholerae. The presented synthetic biology work provides the basis to use secondary chromosomes in E. coli to answer basic research questions as well as for several biotechnological applications.

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“…Detection of a 2‐fold difference in fluorescence however, was near the detection limit of the method; compare distributions for F and an R1‐derived plasmids, the latter having a copy number of 1.7 times that of F (Figure 2B). Previously, both cell‐cycle‐specific (Keasling et al ., 1992; Koppes, 1992; Cooper and Keasling, 1998) and random replication (Leonard and Helmstetter, 1988; Helmstetter et al ., 1997) has been reported. The data presented here lend support to the latter observations.…”

Section: Discussionmentioning

confidence: 99%

Distribution of minichromosomes in individual Escherichia coli cells: implications for replication control

Løbner-Olesen

1999

The EMBO Journal

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A novel method was devised to measure the number of plasmids in individual Escherichia coli cells. With this method, involving measurement of plasmid-driven expression of the green fluorescent protein gene by flow cytometry, the copy number distribution of a number of different plasmids was measured. Whereas natural plasmids had fairly narrow distributions, minichromosomes, which are plasmids replicating only from a cloned oriC copy, have a wide distribution, suggesting that there is no copy number control for minichromosomes. When the selection pressure (kanamycin concentration) for minichromosomes was increased, the copy number of minichromosomes was also increased. At up to 30 minichromosomes per host chromosome, replication and growth of the host cell was unaffected. This is evidence that there is no negative element for initiation control in oriC and that there is no incompatibility between oriC located on the chromosome and minichromosome. However, higher copy numbers led to integration of the minichromosomes at the chromosomal oriC and to initiation asynchrony of the host chromosome. At a minichromosome copy number of~30, the cell's capacity for synchronous initiation is exceeded and free minichromosomes will compete out the chromosome to yield inviable cells, unless the minichromosomes are incorporated into the chromosome.

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Cycle-specific replication of chromosomal and F-plasmid origins

Cited by 3 publications

References 10 publications

Compatible bacterial plasmids are targeted to independent cellular locations in Escherichia coli

Compatible bacterial plasmids are targeted to independent cellular locations in Escherichia coli

Synthetic secondary chromosomes in Escherichia coli based on the replication origin of chromosome II in Vibrio cholerae

Distribution of minichromosomes in individual Escherichia coli cells: implications for replication control

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