S10-positioning influences <i>D</i>. <i>melanogaster</i> infection-capacity of <i>V</i>. <i>cholerae</i>.

<p>Bacterial load within flies 48h pi with parental (n = 53), S10Tnp-35 (n = 21), S10Tnp-510 (n = 24), S10Tnp-1120 (n = 40), S10TnpC2+479(n = 21), ΔS10Tnp-510 (n = 21), ΔS10Tnp-1120 (n = 15), ΔS10TnpC2+479 (n = 11), S10Md(-510;-1120) (n = 15) or S10Md(-1120;C2+479) (n = 15) strains are shown. When the observed value was 0 CFU/fly points were plotted as 10⁰. Statistical significance was analyzed using Kruskal-Wallis non-parametric tests followed by Dunn’s multiple comparisons using parental as control respectively. n.s. stands for non-significant, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001;****, p<0.0001.</p

S10 dosage and expression diminish in a distance-related manner correlating with GR reduction.

<p><b>(a)</b> Expected trend on S10/ter1 and ori1/S10 ratios according to locus repositioning. Ellipses represent chromosomes. Colored dots depict <i>oriC1</i> and <i>oriC2</i> and termini of Chr1 (<i>ter1</i>) and Chr2 (<i>ter2</i>). Simultaneous replication rounds are shown. An orange arrow represents the S10 locus. The expected trend for ori1/s10 and s10/ter1 is shown by top and bottom triangles. <b>(b)</b> Gene dosage measurements obtained by qPCR in fast-growth conditions. <b>(c)</b> S10 expression normalized to parental strains obtained by RT-qPCR. <b>b</b> and <b>c</b> show the mean and error bars representing 95% CI. Statistical significance was assessed by one-way ANOVA two tailed test and Tukey test for multiple comparisons. n.s. stands for non-significant, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001;****, p<0.0001. <b>(d)</b> S10 dosage (red), expression (green) and % variation of μ (blue) of each bacterial strain were plotted as a function S10 position within the genome measured as % of replichore length. Linear regression is plotted for each variable. Chr2 was overlapped to Chr1 according to cell cycle order. Chromosomes are schematized on the top of the graph.</p

Gene dosage measurements performed by qPCR experiments on the full strain set.

<p>Gene dosage measurements performed by qPCR experiments on the full strain set.</p

Generation of S10Tnp strains.

<p><b>(a)</b> S10 is moved by flanking it by HK022 attL and attR sites. Upon transient expression of phage recombinases a DNA circle containing S10 is excised and <i>bla</i> reporter is reconstituted. Viable carbenicillin resistant (Carb^R) cells are obtained if S10 reintegrates at attB’. <b>(b)</b> The obtained S10Tnp strains: ellipses represent chromosomes while small dots represent origin of replication of Chr1 (<i>oriC1</i>,red) and Chr2 (<i>oriC2</i>, blue). Orange arrows depict S10 position within the genome. The green arrow shows <i>bla</i>. Left panel, picture representative of one of the parental strains from which S10Tnp derivatives were produced using attB’ sites at different positions. Right panel, the derivatives showing S10 relocation are shown. Insets correspond to CLSM images of each strain stained with FM5-95, the white bar represents 5 μm (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005156#sec014" target="_blank">Supporting Information</a>).</p

GR defect is consequence of gene dosage reduction.

<p>S10 dosage effect was quantified by averaging obtained μ for each strain and normalizing it to the value of the parental strain. Results are expressed as percentage of the variation (μ %) with 95% CI showing complementation of S10Tnp-1120 mutant. Values were obtained from 5 experiments using several independently obtained clones. Statistical significance was assessed by one-way ANOVA two-tailed test. Tukey test was performed for multiple comparisons. n.s. stands for non-significant, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001;****, p<0.0001.</p