Additional file 1 of Macromolecular crowding links ribosomal protein gene dosage to growth rate in Vibrio cholerae
Creators: name: Soler-Bistué, Alfonso
name: Aguilar-Pierlé, Sebastián
name: Garcia-Garcerá, Marc
name: Marie-Eve Val
name: Sismeiro, Odile
name: Varet, Hugo
name: Sieira, Rodrigo
name: Krin, Evelyne
name: Skovgaard, Ole
name: Comerci, Diego J.
name: Rocha, Eduardo P. C.
name: Mazel, Didier
Publisher: name: figshare
Year: 2020
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. The most affected movants, display lower GFP production than the Parental strain at the single cell level. FRAP experiments were performed in LB at 37 °C taking a photo every 2 seconds for at least 5 minutes using the Parental-1120 strain (Parental, violet), the S10Tnp-1120 (red) and S10TnpC2+479 (blue) movants. The parental was also tested in presence of chloramphenicol at MIC (+ Cm). a) A representative plot showing the recovery of fluorescence over time in individual cells. b) The percentage of FRAP at the endpoint of the experiment is shown for all cells tested. Mean with 95% CI is shown. Statistical significance was analyzed by Kruskal-Wallis test (p&lt;0.0001). Then Dunn multiple comparison test was made for mean rank obtained for each strain. Letters denote groups being statistically different. Figure S2.rpoA and secY overexpression does not rescue growth rate impairment due to S10 relocation. Effect of empty vector, rpoA or secY expression was quantified by averaging the slope (μ) obtained using 4 biological replicates for each strain in different induction conditions. Results are expressed as the mean μ ± 95% CI. Statistical significance was analyzed using a two-way ANOVA two tailed test and Tukey test for multiple comparisons (p&lt;0.0001). Independently of culture conditions differences are not statistically significant between strains harboring the empty vector, pASB25 or pASB26. Expression was repressed by supplementing culture media with 1% glucose. Induction was achieved adding L-arabinose up to 0.2%. Figure S3. RNA Coverage of Chromosome 1 (Chr1) on the full movant strain set. RNA prepared in exponential phase was Deep-Sequenced as described in Materials and Methods. Reads were mapped along the Chr1 of V. cholerae and normalized against the full sequence volume. The graphs show the coverage as Normalized Expression Values (dotted lines indicate 75, 50 and 25e103 NEV) along both replichores of the replicon in ter1-ori1-ter1 order. Each graph represents one strain: Parental (purple); S10Tnp-35 (cyan); S10Tnp-510 (green); S10Tnp-1120 (red); S10TnpC2+479 (blue). The 400 Kbp flanking ori1 are highlighted in orange. A red arrow indicates the peak corresponding to the S10 locus. The coverage of the ori1 region and the size of the S10 peak lowers with increasing S10-ori1 distance (see Table S3). This was not the case for Chr2 where the transcriptional activity of the ori2 region was similar in all strains. Curiously a small increase of the transcriptional activity of the superintegron [81] was observed in S10Tnp-1120 movant (Fig. S4). Figure S4. RNA Coverage of chromosome 2 (Chr2) on selected strains. RNA prepared in fast-growing conditions was subjected to deep sequencing. Reads were mapped along the Chr2 of V. cholerae. The graphs show Normalized Expression Values along both replichores of the replicon in ter2-ori2-ter2 order of the parental and the most affected strains. Each graph represents the coverage along Chr2 length of Parental (purple), S10Tnp-1120 (red) and S10TnpC2+479 (blue). The superintegron [81] is highlighted in red (SI). Interestingly, SI region is overexpressed in the S10Tnp-1120 movant. Scale from the first base is shown above the graphs. Figure S5. Replication dynamics closely correlates S10 location, dosage, ori1 firing and growth rate. a) The slopes obtained from the MFA analyses (white circles, right axis) and the growth rate (black squares, left axis) of each strain were plotted as a function of the S10 genomic location. b) S10 dosage (black triangles, left axis) and ori1/ter1 (white triangles, right axis) ratio from MFA analyses for each strain were graphed as a function of the S10 positioning. c) S10 dosage (red), ori1/ter1 ratio (green) and growth rate (blue) are plotted as a function the slope obtained for each strain in MFA analyses. Linear regression for each variable is shown in dotted lines. The data used for each graphic can be found in Table S3. The obtained correlations and their statistical significance are described in the main text of the article. Figure S6. S10 relocation produces homogeneously distributed global changes in V. cholerae gene expression. Circos plot of genome-wide expression data from strains S10Tnp-35 (Turquoise), S10Tnp-510 (green), S10Tnp-1120 (red) and S10TnpC2+479 (blue). Upper case represents Chr1 while lower case is Chr2 in ter-ori-ter disposition. The origin of replication of each chromosome is represented as oriC1 and oriC2 respectively. From inside to outside: Sense and antisense V. cholerae genes are depicted as dark orange and orange boxes, respectively. Blue bars represent RNA-seq read counts per gene (scale 1-200,000). Fold-change expression relative to the parental strain is indicated as a green or red solid line indicating fold-expression differences higher than 1.2 or lower than 0.8, respetcively. Dark red dots indicate –log (p-value) of the differential expression analysis. Notably, the abundance of significantly altered genes (red dots) from left to right. Figure S7. Manhattan Pot showing statistically altered functions across the movant strain set. The abscissa corresponds to specific COG within the S10Tnp-510 (green), S10Tnp-1120 (red) and S10TnpC2+479(blue). S10Tnp-35 is not included since very few genes are differentially expressed displaying no altered functions. The purple line indicates statistical significance fixing α in 0.05. Figure S8. The growth rate of the parental strain, S10Tnp-1120 and S10TnpC2+479 was measured using automated growth curves at different NaCl concentrations in rich medium. The mean μ value with SEM of 5 independent experiments is shown. Statistical significance was analyzed by one-way ANOVA two-tailed test. Then Holm-Sidak test was done to compare the means values obtained for each strain. Letters denote groups being statistically different within strains. Differences between strains within each NaCl concentration are denoted as follows: *, p&lt;0.05; **, p&lt;0.01; ***, p&lt;0.001 and n.s. stands for non-significant. Figure S9. The growth rate of the parental strain, S10Tnp-1120 and S10TnpC2+479 was measured using automated growth curves at different concentrations of sucrose in LB. The mean μ value with SD of 4 independent experiments by triplicate is shown. All experiments showed the same trend. Statistical significance was analyzed by two-way ANOVA two-tailed test. Then Holm-Sidak test was done to compare the means values obtained for each strain. Letters denote groups being statistically different within strains. Differences between strains within each sucrose concentration are denoted as follows: *, p&lt;0.05; ****, p&lt;0.0001 and n.s. stands for non-significant. Figure S10. MFA profiles plotted as in Fig. 3b and Fig. 6c. Results for the parental strain (purple), S10Tnp-1120 (red) and the S10TnpC2+479 (blue) movants in LB in presence of 5 gr/L (light) or 20 gr/L (dark) are shown. The statistical analysis of these experiments is shown in Figure 6d. Table S1. Full list of plasmids, bacteria strains used in this study. Table S2. Exponential fit of fluorescence (GFP production) as a function of OD600nm. Data was adjusted to the equation Y=Y0*exp(k*X). Table S3. Quantification of genome wide transcriptional activity. We calculated the read coverage of the 400 Kbp flanking ori1 [78]. Table S4: Slope, S10 dosage and ori1/ter1 ratio obtained from MFA analysis and growth rate of the analyzed strain set. Table S5. Transcriptionally altered genes are shared between movants and regulated in the same manner. The proportion of altered genes that is also found to be regulated in either of the other two movants is shown in the first column (grey). The percentage is shown in parentheses. The two entry table shows that these altered genes were transcriptionally altered in the same way between the movants. We calculated the Pearson Correlation Test for their Log2(FC). The value of the test is shown in green while the corresponding p-value is displayed in orange. Table S6. Altered functions upon S10 relocation. Genes within V. cholerae genome were classified in functional categories using eggNOG database. The table shows the number of genes whose expression is altered in selected functional categories for each movant train and the total genes in the chromosome belonging to each category. The number in parenthesis represents the % with respect the total number of genes. The functions with no genes or with no alterations are not displayed and can be found in Additional file 2, Data Set S3. The total number in the last row also includes functions not displayed.