Additional file 1 of The role of two major nucleoid-associated proteins in Streptomyces, HupA and HupS, in stress survival and gene expression regulation
Creators: name: Strzałka, Agnieszka
name: Mikołajczyk, Jakub
name: Kowalska, Klaudia
name: Skurczyński, Michał
name: Holmes, Neil A.
name: Jakimowicz, Dagmara
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Supplementary Material 1: Fig. S1. Growth and sporulation of ΔhupAΔhupS strain. A. Growth curves of wild type (purple), ΔhupA (red), ΔhupAΔhupS (blue), ΔhupAΔhupS::hupA (orange) strains in liquid YEME/TSB medium. Bold lines represent logistic model while striped lines show calculated half time. B. Microscopic images of spores of wild type S. coelicolor and ΔhupAΔhupS strains from 7-day old colonies grown on SFM solid medium. Scale 5 μm. Fig. S2 Impact of hupA and/or hupS deletion on S. coelicolor growth in the presence of H2O2 A. Growth curves of wild type (purple), ΔhupA (red), ΔhupS (green) and ΔhupAΔhupS (blue) strains cultured in ‘79’ medium supplemented with increasing concentrations of H2O2. Solid lines show the growth curve smoothed by the loess algorithm. B. Growth of wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains on solid SFM medium containing increasing concentrations of H2O2. Pictures were taken after 5 days of incubation in 30°C. Fig. S3 Impact of osmotic stress on the growth of wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains Growth curves of wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains in 30 ml of standard (red) and NaCl supplemented (blue, 0.5 M NaCl) YEME/TSB medium. Vertical lines show growth half-time calculated by logistic model. Fig. S4 Comparison of transcriptomes of ΔhupA, ΔhupS, ΔhupAΔhupS strains during different phases of growth A. Scatterplots showing correlation between ΔhupA, ΔhupS and ΔhupAΔhupS transcriptomes from mid-log phase compared to early stationary phase. Pearson correlation coefficient is shown on each plot. Genes found to be significant in both comparisons (FDR ≤ 0.05, |Log2FC| &gt; 1.5) are shown in red, while genes significant in only one of the comparisons are marked as blue or green, grey dots represent genes not affected by hupA and/or hupS deletions. B. Venn diagrams showing significantly changed genes common for analyzed strains ΔhupA, ΔhupS and ΔhupAΔhupS during mid-log and early stationary phase. Fig. S5 RNA-seq and RT-PCR analysis of gene expression A. Principal component analysis (PCA) of the normalized RNA-seq CPM (Counts Per Million) data of S. coelicolor strains in response to hupA (red), hupS (green) or hupAhupS (blue) deletion during mid-log (circles) or early stationary phase (triangles). B. Correlation between Log2FC values obtained from RNA-seq and RT-PCR for genes: dpsA, sigB, gyrB, fabH3, ecrA1, sIHF, lsr, sco0781, gvpA1 from ΔhupA (red), ΔhupS (green) and ΔhupAΔhupS (blue) strains when compared to the wild type strain. C. Table showing Log2FC values of several genes in strains ΔhupA, ΔhupS and ΔhupAΔhupS when compared to the wild type strain during mid-log and early stationary phase, NA value indicates that FDR value was above the 0.05 threshold. Fig. S6 Genes clusters regulated by HupA and HupS A. Heatmap showing normalized expression (CPM) of ArgR regulon genes for ΔhupA, ΔhupS and ΔhupAΔhupS strains during mid-log and early stationary phase. B. Heatmap showing normalized expression (CPM) of OsdR regulon genes for ΔhupA, ΔhupS and ΔhupAΔhupS strains during mid-log and early stationary phase. A, B Genes marked with diamond were found to be significantly changed in comparison to the wild type strain (FDR ≤ 0.05). Fig. S7 Heatmap showing normalized expression (CPM) of actinorhodin cluster genes for ΔhupA, ΔhupS and ΔhupAΔhupS strains during mid-log and early stationary phase. Genes marked with diamond are those whose expression was found to be significantly altered in comparison to the wild type strain (FDR ≤ 0.05). Fig. S8 Changes of gene expression induced by osmotic stress conditions A. Volcano plots showing modified gene expression in ΔhupA, ΔhupS and ΔhupAΔhupS during mid-log and early stationary phase in medium supplemented with 0.5 M NaCl compared to the wild type strain. For each strain significantly changes genes (FDR ≤ 0.05, |Log2FC| &gt; 1.5) are shown in red. Chosen differentially expressed genes are labelled. B. Venn diagrams showing significantly changed genes common for strains ΔhupA, ΔhupS and ΔhupAΔhupS during mid-log or early stationary phase in medium supplemented with 0.5 M NaCl. C. Heatmap showing normalized expression (CPM) of 150 genes with the lowest FDR value calculated for ΔhupAΔhupS strain and wild type comparison in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains during mid-log and early stationary phase in NaCl supplemented medium (0.5 M NaCl). D. Heatmap showing normalized expression (CPM) of SigE regulon genes for ΔhupA, ΔhupS and ΔhupAΔhupS strains during mid-log and early stationary phase in NaCl supplemented medium (0.5 M NaCl). C, D Genes marked with diamond were found to be significantly changed in comparison to the wild type strain (FDR ≤ 0.05, |Log2FC| &gt; 1.5). Fig. S9 All co-expressed clusters of genes identified based on transcriptional changes in ΔhupA, ΔhupS and ΔhupAΔhupS Results of clust analysis of wild type, ΔhupA, ΔhupS and ΔhupAΔhupS transcriptomes from mid-log (red) and early stationary (blue) growth in normal and NaCl supplemented medium. Fig. S10 Modified expression of the genes within biosynthetic gene clusters, ΔhupA, ΔhupS and ΔhupAΔhupS strains during growth at osmotic stress as compared to the wild type strain A. Heatmap showing normalized expression (CPM) of the red cluster genes in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains during mid-log and early stationary phase in NaCl supplemented medium (0.5 M NaCl). B. Heatmap showing normalized expression (CPM) of the carotenoid cluster genes in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains during mid-log and early stationary phase in NaCl supplemented medium (0.5 M NaCl) C. Heatmap showing normalized expression (CPM) of the ectoine cluster genes in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains during mid-log and early stationary phase in NaCl supplemented medium (0.5 M NaCl) D. Heatmap showing normalized expression (CPM) of the desferrioxamine cluster genes in wild type, ΔhupA, ΔhupS and ΔhupAΔhupS strains during mid-log and early stationary phase in NaCl supplemented medium (0.5 M NaCl) A, B, C, D Genes marked with diamond were found to be significantly changed in comparison to the wild type strain (FDR ≤ 0.05).