Journal: Scientific Reports
Article Title: The coordinated action of RNase III and RNase G controls enolase expression in response to oxygen availability in Escherichia coli
Figure Lengend Snippet: Regulation of Eno expression by RNase III and/or RNase G. (a) Effects of rnc and/or rng deletion on the expression level of eno . Escherichia coli MG1655 strains (WT, Δ rng , Δ rnc , and Δ rnc rng ) were grown in LB medium at 37 °C to mid-log phase and harvested for western blot analysis of Eno, Rng, and Rnc using protein-specific polyclonal antibodies. The expression levels of Eno, Rng, and Rnc were compared by setting those of WT to 1. (b) Independent modulation of Eno expression levels by RNase G and RNase III. Western blotting was performed as described for (a) using Δ rnc rng strains harbouring pPM30, pRNG3, or pRNC3. The expression levels of Eno, Rng, and Rnc were compared by setting those of Δ rnc rng harbouring pPM30 to 1. (c) Effects of rnc and/or rng deletion on the eno mRNA abundance. Total cellular RNA was extracted from cultures grown to an OD 600 of 0.6 using an RNeasy mini prep kit. The number of amplicons of enolase and other rnpB mRNA amplified from the cDNAs of the (left) WT, Δ rnc . Δ rng and Δ rnc rng strains (right) harbouring pPM30, pRNC3, or pRNG3. The eno mRNA expression levels were compared by setting those of WT or Δ rnc rng harbouring pPM30 to 1. PCR products were resolved in an 1.5% agarose gel. (d) Identification of the regulatory DNA region that affected the eno expression levels. Top: Schematic diagram of the eno-cat reporter. Bottom: Effects of RNase G and RNase III expression levels on the degree of chloramphenicol resistance of MG1655 cells. MG1655 WT, Δ rng , and Δ rnc cells harbouring pERS1 were transformed with pPM30, pRNG3 (RNase G), or pRNC3 (RNase III). The transformants were grown in LB containing 1 mM IPTG to an OD 600 of 0.6, diluted, and spotted on LB agar containing 0 (Cm 0) or 75 (Cm 75) μg ml −1 chloramphenicol. For (a,b) , the S1 protein was used as an internal standard to evaluate the amount of cell extract in each lane. For (c) , the rnpB mRNA was used as an internal standard to evaluate the amount of cell extract in each lane. For (a–c) , the data are presented as means ± s. e. m. of at least three independent experiments.
Article Snippet: Total RNA samples were prepared from the cultures using an RNeasy mini prep kit (Qiagen).
Techniques: Expressing, Western Blot, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transformation Assay