Fig 1.
Monitoring of pVCR94 and SGI1 in cells by flow cytometry (FC).
A. Schematic representation of chromosomally integrated SGI1. ORFs are represented by gray arrows. Gene numbers correspond to the last digits of locus tags S0xx in the Genbank accession number AF261825. ORFs of interest in this work are shown in brown. AcaCD binding sites are depicted by green angled arrows. Left (attL) and right (attR) junctions with the chromosome are indicated by black bars at the ends. The position of the complex class 1 integron In104 (multidrug resistance region) is indicated by a black arrowhead. B. Fluorescent reporter genes mNeonGreen (green arrow) and mCherry (red arrow) under the control of the PBAD promoter were inserted in pVCR94 between traGC and eexC, and in SGI1 between S009 and S010. C. Representative flow cytometric scatter plots of green and red fluorescence of E. coli KH95 bearing either pVCR94GreenKn or SGI1RedKn. D. Schematic representation of the labelled elements in the cells. SGI1RedKn is integrated into the chromosome at the 3’ end of trmE.
Fig 2.
Incompatibility between SGI1 and IncC plasmids is trackable by FC.
A. Evolution of the percentage of E. coli KH95 cells bearing SGI1RedKn (SGI1) and pVCR94Green (IncC) and grown over 54 generations in the absence of antibiotics as monitored using FC. Plots represent the mean and standard error of the mean obtained from three independent experiments. ATB is a recovery control culture at G54 in LB with selective pressure for both elements. B. Representative FC density plots of the data presented in panel A mapping the green signal (513 nm) as a function of the red signal (610 nm). C. Representative FC density plots of fluorescence intensity over forward scatter corresponding to data presented in panel B. Color keys are identical in all panels. In panel C, pink and red indicate cells producing low- and high-intensity red fluorescence, respectively.
Fig 3.
Replication of SGI1 in the presence of an IncC plasmid.
A. Representative FC density plots of green and red fluorescence at G0 of cells bearing pVCR94Green (IncC) and SGI1Red (WT), its locked-in Δint mutant and its Δrep and ΔoriV mutants. B. Schematic representation of the cell content in the different mutants of panel A. C. Ethidium bromide-stained 0.8% agarose gel electrophoresis of EcoRI-digested plasmid DNA preparation of the same IncC+ cells bearing SGI1Red, its locked-in Δint mutant or locked-out Δint mutant (pSGI1). Lane 1, locked-in trmE::(SGI1Red Δint::aph); Lane 2, SGI1Red; Lane 3, pSGI1; Lane L, 2-Log DNA ladder. D. Effect of deletions on SGI1 copy number. Quantification by qPCR of the SGI1 copy number as measured by the sgiA/chromosome ratios at G0. E. Effect of deletions on SGI1 excision. Quantification by qPCR of the percentage of cells at G0 that contain excised SGI1 as a measure of unoccupied attB sites per 100 chromosomes. “x” indicates that excision was below the limit of detection (<10−5%). Assays were carried out in E. coli KH95 carrying (+) or lacking (-) the helper IncC plasmid pVCR94Green or pVCR94GreenSp (for pSGI1 only). The bars represent the mean and standard error of the mean obtained from at least three independent experiments. Statistical analyses were carried out on the values using the one-way ANOVA with Tukey’s multiple comparison test. F. Effect of SGI1 replication on conjugative transfer of SGI1 and its helper plasmid. Conjugation assays were carried out using E. coli KH95 as the donor and E. coli VB113 as the recipient. When indicated (+), Rep was provided in trans from prep. Transfer frequencies are expressed as the number of transconjugant per donor CFUs. Statistical analyses were carried out on the logarithm of the values using the one-way ANOVA with Sidak’s post-test to compare each mutant set relatively to SGI1Red (WT) control. The bars represent the mean and standard deviation values obtained from at least three independent experiments. Statistical significance is indicated as follow: ****, P<0.0001; ***, P<0.001; *, P<0.05; ns, not significant.
Fig 4.
Identification of the oriV locus of SGI1.
A. Schematic map of the xis-S003 intergenic region of SGI1 containing oriV. B. Sequence of the oriV region of SGI1. Sequence features are indicated and color-coded as in panel A. The stem-loop of the putative rho-independent transcriptional terminator located downstream of S003 has a calculated free energy (ΔG) of -8.9 kcal/mol (ARNold). C. Schematic representation of the S004-rep locus and translational rep’-’lacZ fusion. The translational lacZ fusion was introduced at position 3,246 after the fifth codon of rep (refer to S6A Fig for details). The relative positions of reverse transcription primer rep_RT as well as PCR primers used to amplify rep and S004 fragments are indicated. The dotted line shows reverse transcription product. D. rep expression is controlled by AcaCD. β-galactosidase assays of the translational rep’-’lacZ fusion in SGI1 Δxis performed in IncC-free cells (-), and in the presence of pVCR94 or pacaCD without (no ara) or with arabinose (+ ara). E. Analysis on a 1.5% agarose gel from an assay to amplify rep and S004 on the rep_RT cDNA. Genomic DNA (gDNA) and RNA samples in the absence of reverse transcriptase (noRT) were used, respectively, as positive and negative PCR controls.
Fig 5.
Impact of SGI1 replication on incompatibility.
Evolution of the percentage of E. coli KH95 cells bearing either pVCR94GreenSp (IncC) and pSGI1 (A) or pVCR94Green (IncC) and the ΔtraNS (B), Δint (C), Δxis (D), Δrep (E), or ΔoriV (F) mutants of SGI1Red (SGI1) over 54 generations in the absence of antibiotics as monitored by FC. Inserts for panels C, D and E show complementation experiments using the designated expression plasmid and pVCR94GreenSp. Conditions were identical to Fig 2A. Plots represent the mean and standard error of the mean obtained from three independent experiments. Representative density plots of mNeongreen intensity over mCherry intensity and their corresponding plots of fluorescence intensity over forward scatter are shown for each condition in S1–S5 and S7 Figs.
Fig 6.
Comparison of the genetic maps of 4 SGI1-like genomic islands.
Genomic islands are drawn to scale. The left and right junctions (attL and attR) within the host chromosome are indicated by black bars at the ends. ORFs with similar function are color coded as indicated in the figure. Green flags indicate the position and orientation of AcaCD binding sites predicted using MAST of the MEME suite [30]. Homologous regions are bracketed and linked by a gray line with the corresponding percentage of nucleotide identity. Genbank accession numbers: SGI1, AF261825.2; GI-15, AAWE01000022); GIVchO27-1, CP010812; GISen-26, QDTO01000013.
Table 1.
E. coli K-12 derivative strains, plasmids and genomic islands used in this study.