Figure 8

Transposase- lacZ expression assays in cells over-expressing sRNAs. (A) Transposase expression from an IS50 translational fusion (TLF) (see Figure 3A) present on a low-copy plasmid (pDH798) was measured in the presence of a compatible plasmid expressing one of the indicated sRNAs from the inducible pLlacO promoter in DBH33. Cells were grown in M9 glucose and 0.1 mM IPTG was added to subcultures to induce sRNA expression. Transposase expression was measured 4 hours after IPTG addition. Expression levels were normalized to the strain with the vector only control. (B) The impact of different growth media on SgrS-induced up-regulation of transposase expression was evaluated using a single-copy TCF fusion (see Figure 3A) present in the chromosome of DBH265. Note that the sgrS 1 allele of SgrS contains a two-nucleotide mutation that inhibits its ability to down-regulate expression of the glucose transporter encoded by ptsG. Subcultures were grown in either M9 glucose, M9 glucose + glycerol, or Luria broth (LB), as indicated. β-galactosidase activity was measured approximately 4 to 6 hours after subcultures were started. In (A) and (B) mean and standard error values of duplicate experiments, each of which included at least three replicates, are shown. (C) Northern blot of RNA isolated from cells in (B). RNA was extracted from cells immediately before starting the Miller assay and visualized by Northern blotting with ³²P-labeled RNA probes complementary to either SgrS or the 5S rRNA (internal control).