Fig. 4: The regulatory RNA, sRNA275 (vigR 3′UTR), is required for intermediate-vancomycin tolerance in S. aureus JKD6008. | Nature Communications

Fig. 4: The regulatory RNA, sRNA275 (vigR 3′UTR), is required for intermediate-vancomycin tolerance in S. aureus JKD6008.

From: RNase III-CLASH of multi-drug resistant Staphylococcus aureus reveals a regulatory mRNA 3′UTR required for intermediate vancomycin resistance

Fig. 4

a Spotted dilution assays quantified vancomycin tolerance in sRNA induced CRISPRi knockdown strains. Small RNAs targeted by CRISPRi sgRNAs are indicated above. Culture dilution is indicated left. The cultures were grown in the absence (i) or presence (ii) of 3 μg/mL vancomycin. b Growth curves of VISA (JKD6008), VSSA (JKD6009), pSD1 (vector only control, JKD6008 pSD1), and pSD1-vigR3′UTR (JKD6008 vigR 3′UTR CRISPRi knockdown). Cultures were grown in MH media with or without sub-inhibitory vancomycin (3 μg/ml). c Mapping vigR mRNA 5′ and 3′ ends. Plots indicate raw read data mapping to vigR (E0E12_RS09390) generated using Term-seq (top), or dRNA-seq with (middle) or without (bottom) terminator exonuclease (TEX). The position of the E0E12_RS09390 CDS is indicated below. d Northern blot analysis of vigR mRNA. Total RNA was extracted from strains indicated (top) and probed for vigR RNA. Excepting VSSA (JKD6009), all strains are derived from JKD6008. Sybr Green II stained 23S and 16S rRNAs are indicated below as a loading control. Quantification of the ratio of 16S rRNA to vigR by densitometry is indicated below. e Growth curves for VSSA (JKD6009), VISA (JKD6008), JKD6008 vigR∆3’UTR, JKD6008 vigR∆3’UTR-repair, and JKD6008 vigR∆CDS mutants. Strains were grown in MH media supplemented with antibiotics indicated above.

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