FIGURE 2.

(A) Transcriptional activation of yefM/yoeB loci during amino acid starvation. Exponentially growing (0.45 of OD₄₅₀) cultures of MG1655, Δlon, Δppkppx, and ΔrelAΔspoT were treated with 1 mg/ml of serine hydroxymate. Total RNA was isolated at 0, 10, 30, and 60 min and semi-quantitative primer extension was performed using YefM mRNA-specific primer (YefMPE-2). (B) yefM/yoeB transcriptional activation during overexpression of Lon protease. MG1655 and Δppkppx strains were transformed with pBAD33 vectro or its derivative carrying lon gene. Overnight cultures were diluted and grown to 0.45 OD₄₅₀ in LB medium supplemented with glycerol as carbon source at 37°C. Lon overexpression was induced by addition of 0.2% arabinose. Samples were collected at indicated time intervals and semi-quantitative primer extension performed as described in Materials and methods. (C) YoeB-dependent cleavage upon overexpression of lon is independent of polyP. MG1655, ΔppkΔppx (Δppkx), ΔyefM/yoeB, and Δ5 strains were transformed with pBAD-lon and pBAD-ppk was transformed into MG1655, Δlon, ΔyefM/yoeB and Δ5. The transformants were grown in LB media supplemented with 2% glycerol to mid-exponential phase (0.45 of OD₄₅₀). 0.2% arabinose was added to induce expression of lon or ppk. Samples were collected at 0, 10, 30, and 60 min and primer extension was carried out using Lpp mRNA-specific primer (lpp21) for cleavage site mapping. YoeB-dependent cleavage, indicated by an arrow, is in accordance with results from Christensen et al. (2004).