Table 1.
Primers used in this study.
Fig 1.
Plasmid pSEA1 from Vibrio ponticus.
(A) Circular genetic map of pSEA1. Symbols on the circle are as follows (outside to inside): coordinates, protein-coding sequences coded clockwise, protein-coding sequences coded anticlockwise, G+C content, G+C skew. The Tn6283 region is indicated by arc. Color codes of the coding sequences are as follows: red, partition; blue, site-specific recombination or transposition; light blue, conjugative transfer; green, transposase; orange, antibiotic resistance; yellow, other function; grey, unknown functions. (B) Unrooted phylogenetic tree of MOBH family mobile genetic elements. The tree was constructed based on the maximum-likelihood method using the JTT+G model with PhyML [23].
Fig 2.
(A) Genetic map of Tn6283. The color code for coding sequences is the same as shown in Fig 1. Thioredoxin-dependent thiol peroxidase, a homolog of the product of bcp, is shown in pink. (B) Variation of the pSEA1 integration pattern among transconjugant chromosomes. (i) Restriction map of the boundary between bcp and the left end of Tn6283, and the boundary between the pSEA1 backbone and the left end of Tn6283. The E. coli chromosome is shown as a solid line. The pSEA1 backbone is shown as a dashed line. The scale bar indicates the base pair position. The position at 20 kb upstream of the Tn6283 left-end was set as position 1. (ii) Southern blots for digested genomic DNA. The intA region (indicated by horizontal line) was used as a probe. (C) Integration pattern of Tn6283. The yellow pentagon indicates Tn6283. Colored symbols are the split targets of Tn6283 in pSEA1 (light blue), E. coli chromosome (gray), or V. ponticus chromosome (green). (D) Nucleotide sequences around the Tn6283 insertion sites. The attTn6283 spacer region is boxed. The nucleotide sequence moving with Tn6283 is shown in orange. The nucleotide sequence of the central part of attpSEA1 on the ancestral pSEA1 is not known, and is therefore labeled as "n".
Fig 3.
Interplay of a non-conjugative integrative element and a conjugative plasmid in interspecies antibiotic resistance gene transfer.
(A) Two-step gene transfer model. First, the non-conjugative integrative element is transferred to a recipient cell via suicidal plasmid transfer and is then excised and integrated into the recipient chromosome. The subsequent plasmid transfer allows for integration of the plasmid backbone carrying antibiotic resistance genes into the recipient chromosome via homologous recombination using the homology of the integrative element. The diagram follows the canonical ICE excision model. (B) RecA-dependence of ARG transfer from V. ponticus to E. coli. The data were obtained from four independent mating assays. (C) Tn6283-dependence of ARG transfer from V. ponticus to E. coli. The data were obtained from five independent mating assays.
Fig 4.
Excision of Tn6283 from the E. coli chromosome.
(A) Design for PCR detection of recombination products. The diagrams show the hypothetical scenario in which Tn6283 excises itself as a circular molecule and forms a heteroduplex joint, while the Tn6283 donor site also forms a heteroduplex joint. The PCR-amplified heteroduplex joints should contain two types of sequences in the spacer between terminal repeat sequences. (B) PCR detection of joint formation on the recombination products. Lane 1: long PCR designed to detect the occupied Tn6283 donor site (primer set 2599572R-2599370F). Lane 2: detection of unoccupied Tn6283 donor sites (primer set 2599572R-2599370F). Lane 3: detection of circularized Tn6283 (primer set 3F-3R). (C) Sequences of PCR-amplified joints on the circularized Tn6283. The observed frequency is indicated next to each sequence. (D) Sequences of PCR-amplified joints on the unoccupied Tn6283 donor sites. In the upper panel, two types of expected joint sequences are shown. The lower panel shows the unexpectedly observed sequence, designated Scar.
Fig 5.
Tn6283 excision barely generates unoccupied donor sites in vivo.
(A) A model for Tn6283 excision in E. coli strain LN5. Three models are proposed (see main text for details). Primer sets used for qPCR are indicated as arrows. (B). A model for Tn6283 excision in V. ponticus. (C) Copy numbers of recombination joints in E. coli. Copy numbers of two recombination joints (attTn6283, attB*) shown as the ratio of the number of joints to the number of replicon backbone (dxs) or total Tn6283 molecule (intA). In one sample, the copy number of attB* was below the detection limit. (D) Copy numbers of recombination joints in V. ponticus. Copy numbers of two recombination joints (attTn6283, Scar1) shown as the ratio of the number of joints to the number of pSEA1 backbone (traI) or total Tn6283 molecules (intA). Each dot indicates total DNA extracted from one batch of stationary-phase cultures. The amount of Scar1 was too low to detect in a quantitative manner (outside the range of standard curve), and is thus shown as the numbers below the detection limit (red line: 10−7).
Fig 6.
Tn6283 does not impose a detectable fitness cost on the recipient host.
(A) Growth curve of three E. coli strains cultured in LB. Left: culture OD over 15 hr. The data points represent the means from 16 growth curves. Center: box plot showing the maximum growth rate. Right: box plot showing the maximum OD values. Data were compared among three groups using ANOVA and Tukey’s post-hoc test. (B) Colony-forming units of stationary-phase cultures. Data were obtained from cultures grown in eight distinct wells in 96-well microtiter plates. (C) Cell morphology at three different growth phases. Left: exponential phase (equivalent to 4 hr in the growth curves shown in (A)). Middle: early stationary phase (approximately 7 hr). Right: late stationary phase (20 hr incubation in a microtiter plate). Cells were stained with DAPI (blue) and SYTOX-Green (red). (D) Fraction of dead cells in the exponential phase. Cells in the exponential phase were stained with SYTOX-Green without fixation, and then fluorescence was detected using flow cytometry. The data were filtered to select only single cells using FlowJo software. The data were compared among three groups using the Steels-Dwass test.
Fig 7.
Models for Tn6283 excision pathway.
(Left) Canonical ICE excision. Red: inactive tyrosine recombinase protomer; green, active recombinase protomer. Two rounds of paired strand transfer separate the integrative element and replicon backbone. (Middle) Conservative transposition. Strand transfer occurs on only one strand upon every strand cleavage reaction. This leads to a double-strand break of the donor replicon, similar to the transposition of IS10 and Tn7 [60, 61]. (Right) Copy-out-paste-in transposition. Strand synthesis after the first strand transfer excises the integrative element without cleaving one strand of the parental molecule. Progression of the replication fork generates the circular form of the integrative element, leaving its original copy in the donor molecule.