FIG 5.

STnc540 target identification screen. (a) Pulse expression of STnc540 under SPI-2 conditions. Either one of the two STnc540 isoforms was overexpressed for 10 min prior to total RNA isolation and sequencing. The log₂ fold changes in expression upon induction of the long (x axis) or short (y axis) STnc540 isoform compared to an isogenic strain carrying the empty control vector are plotted. The results represent the means from two biological replicate experiments. mRNAs whose expression was significantly altered (FDR < 0.05) upon induction of at least one STnc540 isoform compared to the control are labeled. SL1344_2328 is a low-confidence target (as inferred from manual read coverage inspection; not shown). (b) Read coverage plot of the expression of the mgt locus upon pulse expression of the long (dark blue) or short (light blue) STnc540 isoform relative to the empty control vector. The position of predicted interaction sites with STnc540 (Fig. S6d) are denoted. TSS annotations (black arrows) were derived from reference 64. (c) Western blot analysis indicates a slight repression of MgtB protein levels upon constitutive overexpression of STnc540 in the presence of ProQ. Wild-type, ΔSTnc540, ΔproQ, or ΔSTnc540 ΔproQ Salmonella with 3xFLAG-tagged MgtB expressed from its native promoter and harboring the indicated, constitutive sRNA overexpression plasmids or pZE-ProQ, respectively, were grown under SPI-2-inducing conditions to an OD₆₀₀ of 0.5. Total protein samples were harvested and analyzed by Western blotting using FLAG-specific or endogenous ProQ-detecting antibodies (the position of ProQ is indicated by an arrowhead; the asterisk denotes an unspecific signal). GroEL serves as a loading control. A representative Western blot experiment out of three independent biological replicates is shown, and the quantification of the MgtB signal intensity in the different strains (normalized to GroEL and relative to the wild-type strain) over the three replicates is given in the graph below the Western blot. (d) Schematic representation of the mgtB::gfp reporter construct used in panel f. The predicted interaction sites with STnc540 (#1 to #4; see Fig. S6d) are indicated. (e) GFP reporter assay for STnc540-dependent regulation of mgtB. The reporter construct in panel d was cotransformed with a constitutive STnc540 (short isoform) overexpression plasmid or the respective vector control into a ΔSTnc540 (top) or ΔSTnc540 ΔproQ (bottom) Salmonella background. The resulting strains were grown under SPI-2-inducing conditions in a 96-well plate for 24 h, and the GFP intensity (as a proxy for MgtB levels) was monitored in 10-min intervals. The data show the means ± SD of the GFP intensity normalized to OD₅₉₅ values and relative to the GFP intensity of the same strains harboring a constitutive GFP expression control plasmid (pXG-1) from three biological replicates, each comprising technical triplicates. In the first 3-4 h, OD, but not GFP intensity, increased (grey windows).