Escherichia coli with a linear genome - PubMed (original) (raw)
Comparative Study
Escherichia coli with a linear genome
Tailin Cui et al. EMBO Rep. 2007 Feb.
Abstract
Chromosomes in eukaryotes are linear, whereas those of most, but not all, prokaryotes are circular. To explore the effects of possessing a linear genome on prokaryotic cells, we linearized the Escherichia coli genome using the lysogenic lambda-like phage N15. Linear genome E. coli were viable and their genome structure was stable. There were no appreciable differences between cells with linear or circular genomes in growth rates, cell and nucleoid morphologies, genome-wide gene expression (with a few exceptions), and DNA gyrase- and topoisomerase IV-dependent growth. However, under dif-defective conditions, only cells with a circular genome developed an abnormal phenotype. Microscopy indicated that the ends of the linear genome, but not the circular genome, were separated and located at each end of a new-born cell. When tos - the cis-element required for linearization - was inserted into different chromosomal sites, those strains with the genome termini that were more remote from dif showed greater growth deficiencies.
Figures
Figure 1
Linearization process of a circular Escherichia coli chromosome mediated by the TelN protein. (A) The circle indicates the E. coli circular genome, in which oriC (replication origin) and the dif site are shown. The two arrows indicate bidirectional replication. (B) Expansion of the dif site from (A). The positions of the dif, tos and kan genes are shown as closed circles, grey and white boxes, respectively. The locations of the two probes used in hybridization experiments and the pair of primers used for the genome linearity PCR assay are shown beneath. The tos site (392 bp) shown in (B) is expanded in (C), and a part of tos (telRL: 56 bp) is expanded in (D). The almost perfect palindromic sequences are shown as a pair of dotted arrows in (D). TelN protein binds to the telRL site and cuts at the staggered positions indicated by vertical arrows in (D), producing two separate ends with long single-stranded regions, each of which can be self-annealed (E), and TelN seals the nicks, producing two ends with hairpin structure (F).
Figure 2
Linearization of the Escherichia coli genome, stability of the linearized genome and growth of the linear genome strain. Linearization assay using agarose gel electrophoresis under neutral and alkaline conditions. (A) Linearization assay using Southern hybridization of chromosomal DNA of MG1655(tos) and its derivatives extracted from overnight cultures, digested with _Sph_I and separated by agarose gel electrophoresis under neutral (a–d) and alkaline (e–h) conditions. Probes were a mixture of probe 1 and 2 shown in Fig 1B. (a,e) MG(tos) in arabinose, (b,f) MG(tos)(pBAD24) in arabinose, (c,g) MG(tos)(pBAD24-telN) in arabinose and (d,h) MG(tos)(pBAD24-telN) in glucose. The positions of _Hind_III-digested λ phage markers are shown to the right and left of the gels. (B) Stability of the linear genome in the MG1655(tos)(N15) strain. Southern hybridizations were carried out after pulse-field gel electrophoresis of agarose-embedded cells grown from a single colony using a mixture of probes 1 and 2 shown in Fig 1B. (a–f) Ethidium bromide staining and (g–l) Southern hybridization patterns. (a,g) A parental MG1655 clone (30 generations), (b,h) an MG (tos)(N15) clone (30 generations), (c,d,i,j) two independent MG (tos)(N15) clones (84 generations) and (e,f,k,l) two independent clones (165 generations). (C) Linear genome strain growth curves. Overnight cultures (0.1 ml) of MG1655, MG1655 (tos) and MG1655(tos)(N15) were inoculated into fresh LB broth (10 ml) and shaken at 37°C; their optical densities (ODs) were measured by spectrophotometer at 770 nm at different time intervals.
Figure 3
Effect of dif deficiency on the linear genome strains. Four different dif_-defective MG1655 derivatives were constructed and their growth rates and cell morphologies were compared. (A) Growth curves were produced as in Fig 2C. Cell morphologies of the parental and the four derivative strains are shown: (B) MG1655, (C) MGΔ_dif, (D) MGΔ_dif_(tos), (E) MGΔ_dif_(N15) and (F) MGΔ_dif_(tos)(N15). OD, optical density.
Figure 4
Relative positioning of the two termini of the Escherichia coli linear genome in vivo. For details on the strains and experiments, see text and Methods. (A) Strain MKG296 (circular genome). Relative positioning of oriC (green) and ter1 (red, −20 kb from dif) can be seen. (B) Strain MK297 (circular genome). Relative positioning of ter1 (red) and ter2 (green, +20 kb from dif) can be seen. (C) Strain MK305 (linear genome). Relative positioning of ter1 (red) and ter2 (green) can be seen. (D) Summary table of relative positions of the two foci in each strain. All cells (126 cells of MKG296; 234 cells of MKG297; 189 cells of MKG305 were used) were classified into the following three patterns: two foci are (i) separated, (ii) close or overlapping, or (iii) have other patterns. The percentage of each pattern and the typical principal patterns are shown in the table. oriC, replication origin.
Figure 5
Terminus location-dependent growth of strains with a linear genome. (A) The circle represents a 0–100 min chromosome map, on which two cis sites, dif and oriC, are shown inside. The other six tos insertion sites, 94 min, 7 min, 20 min, −200 kb, −3 kb (the original linear genome strain) and +200 kb from the dif site, are also shown outside. (B) We inserted the tos_-Km fragment into different sites of the chromosome of MG1655 (or MG) shown in (A), isolated their N15 lysogens and measured their growth as follows: 100 μl of overnight culture of each strain was inoculated into 10 ml of fresh L broth, shaken at 37°C and growth measured spectroscopically at OD600 nm. MG1655(wild type) (open circles); MG(−3 kb∷_tos)(N15) (filled circles); MG(−200 kb∷tos)(N15) (open triangles); MG(+200 kb∷tos)(N15) (open squares); MG(+200 kb∷tos)(tus −)(N15) (filled triangles); MG(7 min∷tos)(tus −)(N15) (horizontal bars); MG(20 min∷tos)(tus −) (N15) (filled squares). OD, optical density; oriC, replication origin.
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