Supplementary figures and tables [Dataset]

<b>Table A.</b> Bacterial strains used in this study. <b>Table B.</b> Composition of CDM and mCDM media used in this study. <b>Table C.</b> Plasmids used in this study. <b>Table D.</b> Primers used in this study. <b>Fig A.</b> Dynamic range of the aTc-inducible CRISPRi system in <i>H. influenzae</i>. (A) Growth of <i>H. influenzae</i> WT (triangles) and <i>dcas9</i> (circles) strains in sBHI. (B) P_tet responsiveness experiment: <i>dcas9</i> strains carrying no (-) or sgRNAs targeting the <i>sspA</i>, <i>fabH</i> or <i>glyA</i> genes were grown for 12 h at 37°C in 10 mL sBHI with shaking (120 r.p.m). Next, cultures were diluted in fresh sBHI to OD₆₀₀ = 0.01 and transferred to a 96-wells plate with a range of aTc concentrations from 0.25 to 50 ng/mL. The color-coded shadow area represents the standard deviation to the mean (solid line). <b>Fig B.</b> Frequency histogram of the <i>E. coli</i> and <i>H. influenzae</i> sgRNA library distribution. sgRNA library distribution in <i>E. coli</i> (A) and in <i>H. influenzae</i> (B). <b>Fig C.</b> sgRNA content variation during CRISPRi screen in sBHI and CDM. (A) Principal component analysis (PCA) shows the disparity between replicates, induction (+ or – aTc), media (sBHI, CDM) and timepoints (7, 14 and 21 generations). + signs represent samples where CRISPRi was activated by aTc; - signs show samples where CRISPRi was uninduced. Blue and red signs represent CRISPRi libraries grown in sBHI and CDM, respectively. Increasing the induction time explains most of the variance in sample strain composition (91%). (B) Clustered heatmap of sgRNA content between replicates, induction (+ or – aTc), media (sBHI, CDM) and timepoints (7, 14 and 21 generations). The normalized read count is expressed in a gradient logarithmic scale. The horizontal axis displays the 1,773 sgRNAs designed for the RdKW20 CRISPRi library, while the vertical axis shows the 48 different samples. <b>Fig D.</b> CRISPRi-based analysis of <i>H. influenzae</i> specific gene fitness in sBHI. <i>dcas9</i> derivative strains expressing <i>infC</i>, <i>rpsL</i>, <i>ispE</i>, <i>metK</i>, <i>rpoC</i>, <i>lpxB</i>, <i>parE, rsxA</i>, <i>rpsT</i> and <i>mepA</i> sgRNAs were grown in sBHI, in the absence (white circles)/presence (black circles) of aTc. Strains were grown in 96-well plates, OD₆₀₀ was measured every 30 min for 15 h; standard deviation to the mean is shown for each timepoint. <b>Fig E.</b> CRISPRi-based analysis of <i>H. influenzae</i> specific gene fitness in CDM. <i>dcas9</i> derivative strains expressing <i>infC</i>, <i>rpsL</i>, <i>ispE</i>, <i>metK</i>, <i>rpoC</i>, <i>lpxB</i>, <i>parE,</i> and <i>rsxA</i>, <i>rpsT</i> and <i>mepA</i> sgRNAs were grown in CDM, in the absence (white circles)/presence (black circles) of aTc. Strains were grown in 96-well plates, OD₆₀₀ was measured every 30 min for 15 h; standard deviation to the mean is shown for each timepoint. <b>Fig F.</b> Pathway enrichment in sBHI. KEGG pathway enrichment analysis in sBHI (<i>versus</i> CDM). The gene ratio refers to the number of enriched genes in sBHI for a specific pathway reported to the total amount of differentially essential genes. Dot size reflects the number of differentially essential genes. Color shades scale the significance (Padj). <b>Fig G.</b> Metabolic map of differentially essential gene enrichment in sBHI. KEGG map (hin01100) of the metabolic landscape in <i>H. influenzae</i> RdKW20 (green). The nodes represent metabolic compounds, and edges represent genes involved in the reactions. Pathways with enrichment in differentially essential genes are framed in orange. Metabolic enzymes differentially more essential in sBHI (<i>versus</i> CDM) are highlighted in red. <b>Fig H.</b> Metabolic map of differentially essential gene enrichment in CDM. KEGG map of the metabolic landscape in <i>H. influenzae</i> RdKW20 (green). The nodes represent metabolic compounds and edges represent genes involved in the reactions. Pathways with enrichment in differentially essential genes are framed in orange. Metabolic enzymes differentially more essential in CDM (<i>versus</i> sBHI) are highlighted in red. <b>Fig I.</b> Differentially essential genes in the glycine, serine and threonine pathway. Path view mapper visualization of enriched/depleted genes using the KEGG database (hin00260) in CDM <i>versus</i> sBHI for the glycine, serine and threonine metabolism. KEGG reference pathway numbers are replaced by gene names when homologs are encoded in the RdKW20 genome. The log₂FC of fitness scores (from DEseq2 analysis) are depicted with a gradient color scale. Red color indicates a negative log₂FC. <b>Fig J.</b> sgRNA gene silencing reversibility in <i>H. influenzae</i>. (A) The <i>dcas9</i> sgRNA_<i>serA</i> strain was grown in mCDM (left) or mCDM + L-serine 10 mM (right), in the absence (white circles)/presence (black circles) of aTc. (B) RdKW20 WT and Δ<i>serA</i> mutant strains were grown in mCDM in the absence (white circles)/presence (grey circles) of L-serine 10 mM. (DOCX)