Figure 2. ChvR expression is controlled by the ChvI-ChvG TCS

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Figure 2. ChvR expression is controlled by the ChvI-ChvG TCS Figure 2. ChvR expression is controlled by the ChvI-ChvG TCS. (A) ChvR is induced in minimal medium. RNA was collected ... Figure 2. ChvR expression is controlled by the ChvI-ChvG TCS. (A) ChvR is induced in minimal medium. RNA was collected from C. crescentus grown in either PYE or M2G to indicated growth phases (OD<sub>660</sub> of 0.2, 0.5, 1.0 and overnight [o/n]), and ChvR expression was determined by Northern blot analysis. (B) chvR is specific to C. crescentus. Synteny analysis of the genomic locus upstream the recF/gyrB operon between C. crescentus, C. sp. K31 and C. segnis. Conserved regions are marked by gray boxes. (C) Mapping of the transposon insertion site. A transposon mutant with reduced PchvR::lacZ reporter activity was recovered, and the Tn5 insertion site was mapped to chvI (first gene of the chvIG-hprK operon). Flanking genes on the C. crescentus chromosome are indicated in white. (D) Expression of ChvR in minimal medium. ChvR expression was determined by Northern blot analysis of RNA samples collected from C. crescentus cultures grown in minimal M2G medium to mid-exponential phase. ChvR levels were compared between wild-type cells, the chvR deletion strain, the ΔlacA reporter strain, the Tn5 mutant recovered from the transposon screen, a chromosomal deletion of chvIG-hprK, and a complementation strain in which the chvIG-hprK operon is expressed under control of its own promoter from the xyl locus on the C. crescentus chromosome. (E) Expression of ChvR upon DNA damage requires integrity of the chvIG-hprK operon. RNA was collected from C. crescentus grown in PYE 240 min after treatment with MMC, and ChvR levels were determined by Northern blot analysis. (F) Expression of ChvR in acidified growth medium. C. crescentus was grown in PYE to mid-exponential phase when cultures were split, and growth was continued in PYE at either neutral (N; pH 7) or acidic pH (A; pH 5.5). RNA samples were collected at indicated time-points, and ChvR levels were determined by Northern blot analysis. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, Volume 46, Issue 20, 27 August 2018, Pages 10969–10982, https://doi.org/10.1093/nar/gky765 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3. ChvR acts as a repressor of chvT mRNA. (A) C Figure 3. ChvR acts as a repressor of chvT mRNA. (A) C. crescentus ΔchvR cells carrying either plasmid pPvan::ChvR ... Figure 3. ChvR acts as a repressor of chvT mRNA. (A) C. crescentus ΔchvR cells carrying either plasmid pPvan::ChvR (pKF382-1), or a control plasmid (pBVMCS-6) were grown in minimal M2G medium to mid-exponential phase (OD<sub>660</sub> of 0.6) when vanillate was added to induce ChvR expression. RNA samples were collected prior to and at indicated time-points after sRNA induction, and ChvR levels were determined by Northern blot analysis. (B) Microarray analysis of C. crescentus genes affected by pulse overexpression of ChvR. Changes in transcript abundances between C. crescentus ΔchvR in response to ChvR overexpression and a control sample were scored on C. crescentus-specific microarrays. Dashed vertical or horizontal lines in the volcano plot indicate cut-off criteria of target selection (>3-fold change; P-value < 0.005), and chvT is marked in blue. (C) Verification of microarray results by qRT-PCR analysis. Abundances of chvT and recF mRNAs were determined in independent RNA samples collected as described in (A). The signal obtained in the control sample was set to 1; error bars represent the standard deviation calculated from two independent biological replicates. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, Volume 46, Issue 20, 27 August 2018, Pages 10969–10982, https://doi.org/10.1093/nar/gky765 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4. ChvR controls ChvT::3XFLAG production under different environmental conditions. (A–C) Samples were collected ... Figure 4. ChvR controls ChvT::3XFLAG production under different environmental conditions. (A–C) Samples were collected from C. crescentus wild-type (carrying a control plasmid (pBXMCS-6)), or a chvR deletion strain (carrying either a control plasmid, or a multi-copy construct expressing chvR from its own promoter (pKF370-1)). In all strains, chvT is marked by a C-terminal 3XFLAG affinity tag. Protein and RNA levels were determined by Western blot and Northern blot analysis, respectively. (A) C. crescentus cells were grown in minimal M2G medium to indicated growth phases (OD<sub>660</sub> of 0.2, 0.5, 1.0 and overnight [o/n]). (B) Expression of ChvR and ChvT::3XFLAG was determined in C. crescentus grown in PYE prior to (OD<sub>660</sub> of 0.5; [-]), and 240 min after treatment with MMC [+]. (C) C. crescentus was grown in PYE to mid-exponential phase (OD<sub>660</sub> of 0.5) when cultures were split, and growth was continued in PYE at either neutral (N; pH 7) or acidic pH (A; pH 5.5). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, Volume 46, Issue 20, 27 August 2018, Pages 10969–10982, https://doi.org/10.1093/nar/gky765 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 1. Transcriptome analysis of C Figure 1. Transcriptome analysis of C. crescentus in response to DNA damage. (A) Diagram summarizing the results of the ... Figure 1. Transcriptome analysis of C. crescentus in response to DNA damage. (A) Diagram summarizing the results of the dRNA-seq experiment. RNA was collected from C. crescentus grown in PYE prior to (OD<sub>660</sub> of 0.4) or 240 min after treatment with the DNA-damaging agent MMC. Under these conditions, 169 genes were induced >3-fold, including 9 sRNAs. No conserved SOS box was identified in proximity to transcriptional start sites of sRNAs. (B) Expression of ChvR sRNA in wild-type C. crescentus. Cells were grown in PYE, and RNA samples were collected prior to (OD<sub>660</sub> of 0.4) and at indicated time-points after MMC treatment, or at different time-points over growth (OD<sub>660</sub> of 0.2, 0.5, 1.0, 3h after cells had reached OD<sub>660</sub> of 1.0, and overnight [on]), respectively. ChvR levels were determined by Northern blot analysis; 5S rRNA served as loading control. (C) cDNA reads of +/- MMC libraries mapping to the chvR/recF locus of C. crescentus NA1000. Libraries established from terminator exonuclease (TEX)-treated RNA samples are represented in blue, libraries established from untreated RNA in black, respectively. All libraries were adjusted to the same scale. Annotation and genome position are indicated in the centre. Transcriptional start sites are marked by arrows. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, Volume 46, Issue 20, 27 August 2018, Pages 10969–10982, https://doi.org/10.1093/nar/gky765 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5. ChvR interacts with two independent sites of chvT mRNA Figure 5. ChvR interacts with two independent sites of chvT mRNA. (A) Schematic representation of the translational ... Figure 5. ChvR interacts with two independent sites of chvT mRNA. (A) Schematic representation of the translational chvT::gfp fusions (under control of the constitutive PrsaA promoter). Reporters comprise the 5′ untranslated region plus the first 45 nucleotides of the chvT CDS. Location of ChvR binding sites 1 and 2 are indicated by blue boxes, positions of single nucleotide exchanges are marked in red. (B) Partial repression of chvT::gfp by ChvR via binding site 1. C. crescentus ΔvanAB carrying the indicated gfp reporter fusion were co-transformed with either a control plasmid (pBVMCS6), or a plasmid overexpressing ChvR or ChvR-M1 under the control of the vanillate-inducible promoter Pvan (pKF382-1 or pKF395-1, respectively) were grown overnight in PYE supplemented with vanillate. GFP fluorescence was determined by plate reader measurements. For each fusion, GFP levels in the presence of the control plasmid were set to 1, and relative changes were determined for cells expressing ChvR. GFP levels were calculated from three biological replicates; error bars indicate the standard deviation. (C, D) Secondary structures of ChvR and the 5′ UTR of chvT mRNA (from the transcriptional start site to the start codon) based on bioinformatic predictions (45). (E) Predicted base-pairing interactions forming between ChvR and chvT mRNA. Both interactions at binding site 1 (base-pairing of the second stem-loop of ChvR (nts 31–42) and chvT mRNA (nts −70 to −59 relative to the translational start site)) and at binding site 2 (base-pairing of the third stem-loop of ChvR (nts 63–73) and chvT mRNA (nts −20 to −10 relative to the translational start site). Positions of single-nucleotide exchanges generating the compensatory mutants M1 and M2 are indicated. Expression of all ChvR variants was confirmed by Northern blot analysis (Supplementary Figure S7B). (F-G) Analysis of GFP fluorescence of C. crescentus ΔvanAB carrying the indicated gfp reporter fusion in combination with either a control construct, or plasmids overexpressing ChvR, ChvR-M1, ChvR-M2 (pKF414-1) or ChvR-M1M2 (pKF418-1). Experimental details as in (B). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, Volume 46, Issue 20, 27 August 2018, Pages 10969–10982, https://doi.org/10.1093/nar/gky765 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 6. Post-transcriptional regulation by ChvR is independent of Hfq. (A) Analysis of GFP fluorescence of C. ... Figure 6. Post-transcriptional regulation by ChvR is independent of Hfq. (A) Analysis of GFP fluorescence of C. crescentus ΔvanAB (WT) or ΔvanAB Δhfq (Δhfq) cells carrying the indicated gfp reporter fusion in combination with either a control construct, or a plasmid overexpressing ChvR. Experimental details as in Figure 5 (B). (B) Samples were collected from C. crescentus wild-type (carrying a control plasmid (pBXMCS-6)), or a chvR deletion strain (carrying either a control plasmid, or a multi-copy construct expressing chvR from its own promoter (pKF370-1)), as well as isogenic hfq mutants of these strains grown in minimal M2G medium to an OD<sub>660</sub> of 0.5. In all strains, ChvT is marked by a C-terminal 3XFLAG affinity tag. Protein and RNA levels were determined by Western blot and Northern blot analysis, respectively. (C) Expression of ChvR in an hfq mutant strain. C. crescentus wild-type and Δhfq cells were grown in M2G to indicated time-points over growth (OD<sub>660</sub> of 0.2, 0.5, 1.0 and overnight [on]). RNA expression of ChvR and CCNA_R0014 were determined by Northern blot analysis. (D) ChvR does not interact with Hfq in vivo. C. crescentus wild-type or a 3XFLAG::hfq strain were grown in minimal M2G medium to OD<sub>660</sub> of 1.0, and cell lysates were subjected to immunoprecipitation using a monoclonal anti-FLAG antibody. Protein and RNA samples of lysate (input) and co-immunoprecipitation fractions (co-IP) were analysed by Western and Northern blot analysis, respectively. AB: recovered anti-FLAG antibody. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Res, Volume 46, Issue 20, 27 August 2018, Pages 10969–10982, https://doi.org/10.1093/nar/gky765 The content of this slide may be subject to copyright: please see the slide notes for details.