Figure 1. srn_3610_sprC expression increases in the absence of SarA

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Figure 1. srn_3610_sprC expression increases in the absence of SarA Figure 1. srn_3610_sprC expression increases in the absence of SarA. (A) Srn_3610_sprC expression in different Staphylococcus aureus strains lacking transcription factors (TFs). Northern blot analysis of the Srn_3610_sprC and transfer-messenger RNA (tmRNA) transcripts in the S. aureus HG003 strain and in isogenic mutants for the SarA protein family. All samples were harvested after 5 h of growth. A total of 10 μg of total RNA were analyzed, and the Digoxigenin method (Roche) was used to reveal Srn_3610_sprC and tmRNA (loading control). (B) Northern blot analysis of Srn_3610_SprC and tmRNA transcript expression in S. aureus HG003 and isogenic HG003ΔsarA. Samples were harvested at the specified time point, and tmRNA was used as a control. In a graph showing optical density as a function of time, the growths of HG003 (diamonds) and HG003ΔsarA (triangles) are represented in a semi-logarithmic scale. From: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus Nucleic Acids Res. 2016;44(21):10186-10200. doi:10.1093/nar/gkw777 Nucleic Acids Res | © The Author(s) 2016. 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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 2. SarA represses srn_3610_sprC expression Figure 2. SarA represses srn_3610_sprC expression. (A) Staphylococcus aureus HG003 and HG003ΔsarA strains were co-transformed with: pCN36/pCN41c empty vectors (lanes 1 and 5); pCN36/pCN41c-P<sub>sprC</sub> (lanes 2 and 6); pCN36-SarA/pCN41c (lanes 3 and 7); and pCN36-SarA/pCN41- P<sub>sprC</sub> (lanes 4 and 8). Srn_3610_SprC and SarA transcripts levels were assessed by northern blot. A 16S rRNA probe was used to reflect total RNA loading for each lane. In parallel, SarA protein levels were checked by western blot (middle panel) and SYPRO Ruby staining of total protein extracts was done to compare loaded protein levels (bottom). (B) Effects of SarA on transcriptional activity of the srn_3610_sprC promoter (P<sub>sprC</sub>). S. aureus HG003 wild-type strain (HG003) and a strain lacking sarA (HG003ΔsarA) were co-transformed with pCN41c or pCN41c-P<sub>sprC</sub> and either pCN36 or pCN36-SarA. P<sub>sprC</sub> activity was estimated by measuring ß-lactamase substrate hydrolysis. For each lane, indicated ß-lactamase activity was normalized by subtracting the background signal from the same strain where pCN41c-P<sub>sprC</sub> was replaced by pCN41c empty vector (not shown). Three independent experiments were done, error bars show ± standard deviation. (Mann–Whitney test; *P < 0.05; ***P < 0.001.). From: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus Nucleic Acids Res. 2016;44(21):10186-10200. doi:10.1093/nar/gkw777 Nucleic Acids Res | © The Author(s) 2016. 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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 6. In vivo analysis of the srn_3610_sprC and srn_9340 promoters: SarA versus σ<sup>A</sup> binding. The Staphylococcus aureus HG003ΔsarA strain was transformed either with an empty pCN38 vector (dark gray) or with pCN38-SarA6His (light gray). Chromatin immunoprecipitation (ChIP) experiments were conducted using antibodies for 6His-tag (A) or for the σ<sup>70</sup>/σ<sup>A</sup> RNA polymerase subunit (B). DNA enrichment was assessed by qPCR analysis using specific primers for the promoters of srn_3610_sprC (left panel), srn_9340 (middle) or hla (right). Values beneath the bars indicate fold change between specific and non-specific interactions. (Mann–Whitney U test; three independent experiments; *P < 0.05). (A) srn_3610_sprC, srn_9340 and hla promoters co-immunoprecipitate with SarA6His. (B) SarA6His disturbs σ<sup>A</sup> binding on the srn_3610_sprC promoter. From: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus Nucleic Acids Res. 2016;44(21):10186-10200. doi:10.1093/nar/gkw777 Nucleic Acids Res | © The Author(s) 2016. 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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 5. SarA binds srn_9340 on its promoter and represses transcription. (A) SarA represses srn_9340 expression. After 5 h of growth, 10 μg of total RNA were obtained from Staphylococcus aureus HG003 and HG003ΔsarA. Srn_9340 expression levels were monitored by northern blot. (B) Schematic representation of both the 5΄ and 3΄ ends of the Srn_9340 transcripts (long and short small RNAs), as determined by rapid amplification of cDNA ends (RACE). (C) SarA binds the srn_9340 promoter in vitro. An EMSA was done using 10 fmol of a <sup>32</sup>P-labeled srn_9340 promoter fragment (srn_9340<sub>P225</sub>) as a probe in the presence of increasing amounts (0.01–1 pmol) of 6His-tagged SarA. Srn_9340<sub>P225</sub> forms an initial complex with 0.15 pmol of SarA, and a second one at 0.75 pmol. (D) SarA specifically binds srn_9340<sub>P225</sub>in vitro. EMSA was done with 10 fmol of <sup>32</sup>P-labeled srn_9340<sub>P225</sub>, 1 pmol of 6His-SarA and increasing amounts of specific (unlabeled srn_9340<sub>P225</sub>) or non-specific competitors (unlabeled 16S<sub>225</sub>). (E) DNase I footprinting assays were performed in the presence of 10 fmol srn_9340<sub>P225</sub>, 7.5.10<sup>−2</sup> U DNase I and increasing amounts (0.1–0.5 pmol) of 6His-SarA. The region protected by SarA is indicated with a vertical dotted arrow, and the numbers indicate relative positions to the previously determined transcription start site (TSS). Lanes G, A, T and C correspond to sequencing. The nucleotides from −51 to −1 (srn_9340<sub>51</sub>) are protected by SarA against DNase I degradation. (F) Schematic representation of the DNA probes used for EMSA studies. (G) Deletion of the protected SarA sequence from the srn_9340 promoter region abolishes SarA's capacity to bind the srn_9340 promoter. (H) A 51 bp protected sequence is sufficient for SarA binding. EMSA were done using 10 fmol srn_9340<sub>51</sub> in the presence of increasing amounts of 6His-tagged SarA (0.25–2 pmol). (I) Srn_9340<sub>51</sub> competes with srn_9340<sub>225</sub>for SarA binding. EMSA was performed with 10 fmol of <sup>32</sup>P-labeled srn_9340<sub>P225</sub>, 0.4 or 1 pmol of 6His-SarA and increasing amounts of specific (unlabeled srn_9340<sub>51</sub>) or non-specific competitors (unlabeled Random<sub>51</sub>). (J) The 23 bp SarA binding site on srn_9340<sub>225</sub>was confirmed by EMSA done in the presence of 0.5 pmol sarA and with increasing amounts of unlabeled srn_9340<sub>225</sub>or unlabeled srn_9340<sub>225</sub><sub>Δ23</sub>. From: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus Nucleic Acids Res. 2016;44(21):10186-10200. doi:10.1093/nar/gkw777 Nucleic Acids Res | © The Author(s) 2016. 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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 4. srn_9340 possesses a putative binding site for SarA Figure 4. srn_9340 possesses a putative binding site for SarA. Srn_3610_sprC belongs to the pathogenicity island SaPIn3. SprC<sub>47</sub>, which can interact with SarA, possesses a 22-nt palindromic sequence (underlined). Blast analysis using sprC<sub>47</sub> as the query led to the discovery of another putative SarA binding site within SaPIn3. This sequence was identified as part of the promoter of another small RNA, Srn_9340. From: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus Nucleic Acids Res. 2016;44(21):10186-10200. doi:10.1093/nar/gkw777 Nucleic Acids Res | © The Author(s) 2016. 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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 3. SarA specifically binds a region overlapping the srn_3610_sprC promoter and its 5΄ end. (A) SarA binds the srn_3610_sprC promoter in vitro. Electrophoretic mobility shift assays (EMSA) were done using 10 fmol sprC<sub>P267</sub> probe, a <sup>32</sup>P-labeled srn_3610_sprC promoter fragment and increasing amounts of 0.1 to 8 pmol 6His-tagged SarA. (B) SarA specifically binds sprC<sub>P267</sub>. EMSA was performed with 4 pmol of SarA and with increasing amounts of specific (unlabeled sprC<sub>P267</sub>) or non-specific competitors (unlabeled 16S<sub>267</sub>). The sprC<sub>P267</sub>/SarA complex is only inhibited in the presence of 50–100× excesses of the specific competitor. (C) The SarA binding site overlaps the srn_3610_sprC promoter and gene. DNase I footprinting assays were performed in the presence of 10 fmol sprC<sub>P267</sub>, 2.10<sup>−2</sup> U DNase I, and increasing amounts (0.5–2 pmol) of 6His-SarA. Lanes 1–4 correspond to sprC<sub>P267</sub> sequencing, and the promoter region is annotated. The vertical dotted arrow indicates the srn_3610_sprC region protected from DNase I degradation. The SarA binding site (−32 to +15) was named ‘sprC<sub>47</sub>.’ (D) Schematic representation of the different DNA probes used for EMSA studies. (E) The 47 bp region protected by SarA is necessary for SarA binding with srn_3610_sprC. EMSA were realized as in A, using <sup>32</sup>P-labeled sprC<sub>P267Δ47</sub> (right) or wild-type <sup>32</sup>P-labeled sprC<sub>P267</sub> (left). SarA was unable to form a complex with sprC<sub>P267Δ47</sub>. (F) The 47 bp protected by SarA is sufficient for SarA binding onto srn_3610_sprC. EMSA were done using 10 fmol sprC<sub>47</sub>, 4 pmol of 6His-tagged SarA, and increasing amounts of either unlabeled sprC<sub>47</sub> as a specific competitor or unlabeled 47-bp sequence (Random<sub>47</sub>) as a non-specific competitor. SarA forms a complex with DNA (up-shifted band) that can only be disrupted by an excess of the specific competitor. (G) A 22-bp SarA binding site on sprC<sub>P267</sub> was found with EMSA done in the presence of 0.5 pmol of sarA and with increasing amounts of unlabeled sprC<sub>P267</sub> or unlabeled sprC<sub>P267Δ22</sub>. From: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus Nucleic Acids Res. 2016;44(21):10186-10200. doi:10.1093/nar/gkw777 Nucleic Acids Res | © The Author(s) 2016. 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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 7. SarA and bacterial RNA polymerase holoenzyme cannot simultaneously bind sprC<sub>P267</sub> and srn_9340<sub>P225</sub>. EMSA was done using 10 fmol <sup>32</sup>P-labeled sprC<sub>P267</sub> or srn_9340<sub>P225</sub> probes with increasing amounts (2–100 fmol) of purified RNA polymerase. The binding capacity of Escherichia coli RNA polymerase was confirmed by EMSA experiments using sprC<sub>P267</sub> (A) or srn_9340<sub>P225</sub> (B). To demonstrate their binding exclusivity on sprC<sub>P267</sub> (C) or srn_9340<sub>P225</sub> (D), 0.5 pmol purified SarA and 50–100 fmol E. coli RNA polymerase were simultaneously added to EMSA experiments. In vitro transcription was realized using 10 fmol of sprC<sub>456</sub> (E) or srn_9340<sub>337</sub> (F) and 100 fmol of E. coli RNA polymerase. A total of 5 pmol of the purified TF SarA were added, when indicated. From: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus Nucleic Acids Res. 2016;44(21):10186-10200. doi:10.1093/nar/gkw777 Nucleic Acids Res | © The Author(s) 2016. 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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Figure 8. Analysis of the SarA-protected sequences on the srn_3610_sprC and srn_9340 promoters. The alignment of the sprC<sub>47</sub> and srn_9340<sub>51</sub> sequences was performed using Clustal Omega software (http://www.ebi.ac.uk/Tools/msa/clustalo/). Boxes outline the −35 (yellow) and −10 (red) boxes. The TSS is indicated with a black arrow; the SarA binding site defined by Sterba et al. (5΄ATTTTAT3΄) is shown with dotted arrows (reverse complementary sequences); palindromic sequences are depicted by large arrows; and identical nucleotides are identified with stars. The 22 nt of sprC<sub>47</sub> and the 23 nt of srn_9340 that are necessary for SarA binding are bold. From: Insights into the regulation of small RNA expression: SarA represses the expression of two sRNAs in Staphylococcus aureus Nucleic Acids Res. 2016;44(21):10186-10200. doi:10.1093/nar/gkw777 Nucleic Acids Res | © The Author(s) 2016. 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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com