Replication of the Salmonella Genomic Island 1 (SGI1) triggered by helper IncC conjugative plasmids promotes incompatibility and plasmid loss
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.
