Figure 1.
Probable actions of restriction-modification (RM) systems.
A. Post-segregational killing. Loss or disturbance of an RM system leads to chromosomal breakage by the R enzyme and, unless repaired, to cell death. B. Two possible roles of R in the successful movement. (i) Selection. The RM system on a plasmid moves to a new locus in the genome. The cells with the unrearranged genome are killed by RM attack after the plasmid loss, while the cells carrying the chromosome into which RM is incorporated would survive. (ii) Movement. RM-mediated DNA breakage promotes movement.
Figure 2.
Experimental design for detection of RM gene complex movement.
A. The donor plasmid pSO429, which relies on temperature-sensitive (pSC101-derived) replication machinery, carries the PaeR7I RM gene complex and three drug-resistance genes (Ampr, Kmr and Cmr). B. The plasmid was introduced into E. coli. MG1655 strain. The shift to the temperature non-permissive for replication induce cell death due to post-segregational killing, and would allow growth of the cells carrying the plasmid harboring PaeR7I RM genes.C. In the absence of ampicillin (series 1) or in its presence (series 2), cells retaining at least one drug-resistance gene (Ampr or Kmr) and missing one (series 1), or cells retaining two drug-resistance genes (Ampr and Kanr, or Ampr and Cmr) and missing one (series 2) were selected and screened at 42°C on solid medium. The double drug-resistant clones (three types in series 1 and two types in series 2) obtained from the above process were subjected to sequencing to determine the integration point on the chromosome.
Figure 3.
Detection of PaeR7I RM gene complex movement after replication block.
A. (Series 1) E. coli cells carrying pSO429 (pSC101ts, PaeR7I R+M+, Ampr, Kmr and Cmr) or pSO431 (the R− version) were isolated and grown to log phase at 30°C with the three selective antibiotics. Cells were then centrifuged and re-suspended in LB broth at 42°C without antibiotics (hour 0). After incubation with aeration at 42°C, an aliquot was spread on agar containing antibiotics and incubated overnight at 42°C in order to select and screen for transposition clones as described in Figure 2. Viable cells were calculated from the number of colony formers on LB agar without selective antibiotics after incubation at 30°C. Plasmid-carrying cells were calculated from the number of colony formers on LB agar with selective antibiotics after incubation at 30°C. Transposition frequencies were calculated as (number of double drug-resistant cells per ml)/(number of viable cells per ml). Average values of six independent tubes were plotted. B. (Series 2) E. coli cells carrying pSO429 (R+) or pSO431 (R−) were grown and prepared for the temperature shift as in A. At hour 0, cells were centrifuged and re-suspended in LB broth containing a low dose (25 µg/ml) of Amp at 42°C, and incubated with aeration at 42°C. Aliquots were subjected to selection and screening as shown in Figure 2C and the transposition frequency, as well as the number of viable cells, plasmid-carrying cells were calculated as in A. The averages of 5 (R+) or 4 (R−) measurements were plotted. The upper graphs show relative colony forming unit and the lower graphs show calculated transposition frequency.
Figure 4.
A. Three types of transposition products. (I) Apparent co-integrate. Apparent co-integration of the plasmid and the chromosome at IS1 or IS5 already present on the chromosome. IS1 integration was associated with target duplication (TD in red) of 8–10 bp at various donor (plasmid) sites. IS5 generated a 4-bp target duplication at various donor sites (Figures S2and S3). (II) Aberrant IS insertion. De novo insertion of IS1 with the entire plasmid, except for a 1- or 3-bp terminal deletion (black parentheses). The chromosomal locus had a 9-bp target duplication. The right end of the plasmid insert was similar to the end of IS1 in one product (Figure S4). (III) Reciprocal recombination. Reciprocal crossing-over between the plasmid and the chromosome involving 1- or 3-bp region of sequence identity (Figure S5). B. Hypothetical routes to the products. (a) One-step co-integrate formation between the chromosome and the plasmid at a chromosomal IS. (b) Chromosomal IS insertion into the RM plasmid. (c) Homologous recombination between the plasmid IS and a homologous chromosomal IS leading to the apparent co-integrate. (d) Plasmid IS experiences one-ended transposition into a new chromosomal target site with target duplication. (e) Plasmid and chromosome undergo reciprocal illegitimate recombination at the 1–3 bp identity. Symbols: green, chromosome; white arrow, IS; gray, plasmid; red, plasmid target duplication; black, chromosomal target duplication.
Figure 5.
Reconstruction experiment demonstrating selective advantage of transposition products in an R-positive culture.
One transposition clone from an R-positive (R+) culture and one from an R-negative (R−) culture were labeled with the Tetr gene (materials and methods). Temperature-shift experiment was performed as described in Figure 3B, in the presence of Amp, with Tets cells carrying an R+ (Tube 1) or R− (Tube 4) ts plasmid. At hour 12, about 50/ml of Tetr-labeled transposition cells was added to portions from tube 1 to make tube 2 (Tetr-labeled R+ clone added) and tube 3 (Tetr-labeled R− clone added), and to portions from tube 4 to make tube 5 (Tetr-labeled R+ clone added) and tube 6 (Tetr-labeled R− clone added). All 6 tubes were aerated at 42°C until hour 24. Bulk transposition cells (dotted lines) were measured as colonies that grew on LB agar with Amp (with Oxa) and Cm at 42°C, but not on Kan agar, as for Figure 3B. Tetr-labeled transposition cells (solid lines) were measured as colony formers on LB agar supplemented with Tet at 42°C. The culture was diluted twice to avoid entrance to saturation. The relative concentrations of colony formers in the absence of the dilution were calculated and plotted. The average values from two independent tubes were plotted.