Fig. 3.

DinR is an Hfq-associated sRNA produced in response to DNA damage. (A) RNA samples recovered from the Hfq RIP-seq analysis (Fig. 1) were analyzed on Northern blots and probed with a DinR-specific riboprobe to determine expression and enrichment of dinI mRNA and its processing products including DinR. 5S rRNA served as loading control. (B) Upper: Organization of the dinI locus on the main chromosome of K. pneumoniae MGH 78578. A LexA-box overlapping the dinI TSS is indicated by an orange box. The dinI primary transcript (348 nt) is processed, releasing DinR (157 nt). Lower: Nonredundant alignment of the dinI 3′UTR in diverse enterobacteria (kpn: Klebsiella pneumoniae MGH 78578; ent: Enterobacter sp. 638; eco: E. coli MG1655; sfl: Shigella flexneri 301; cro: Citrobacter rodentium ICC168; stm: Salmonella Typhimurium LT2); the dinI stop codon and the Rho-independent transcriptional termination site are shaded in gray. Nucleotides are colored regarding their degree of conservation (red: high conservation; blue: partial conservation; black: little or no conservation). (C) Expression of dinI mRNA and DinR sRNA in WT and Δhfq K. pneumoniae. RNA samples were collected at different time points over growth (OD₆₀₀ from 0.25 to 2.0, and 3 h after cells had reached OD₆₀₀ of 2.0) and analyzed by Northern blotting. 5S rRNA served as loading control. (D) In vivo binding of LexA to the K. pneumoniae dinI promoter. Association of LexA before and after addition of MMC was determined by ChIP (+AB: anti-LexA antibody; −AB: no antibody control) followed by quantitative PCR. Relative enrichment of DNA fragments was calculated as a ratio between the tested promoter region and a control region located within the sgrR CDS. (E) Expression of DinR in response to DNA damage. WT cells were cultivated in LB to OD₆₀₀ of 2.0. RNA samples were collected prior to and 30 min after induction of DNA damage with MMC, CPX or UV, or from an untreated control (ctrl.). Expression of dinI mRNA and DinR was assessed by Northern blot analysis; 5S rRNA served as loading control. See SI Appendix, Fig. S7 for a relative comparison of dinI mRNA and DinR expression levels.